Novel crispr-cas systems for genome editing

ABSTRACT

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell, using a novel Cas endonuclease. The methods and compositions employ a guide polynucleotide/endonuclease system to provide an effective system for modifying or altering target sequences within the genome of a cell or organism. Also provided are novel effectors and endonuclease systems and elements comprising such systems, such as guide polynucleotide/endonuclease systems comprising an endonuclease. Compositions and methods are also provided for guide polynucleotide/endonuclease systems comprising at least one endonuclease, optionally covalently or non-covalently linked to, or assembled with, at least one additional protein subunit, and for compositions and methods for direct delivery of endonucleases as ribonucleotide proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/779,989 filed 14 Dec. 2018, U.S. Provisional Application No.62/794,427 filed 18 Jan. 2019, U.S. Provisional Application No.62/819,409 filed 15 Mar. 2019, U.S. Provisional Application No.62/852,788 filed 24 May 2019, and 62/913,492 filed 10 Oct. 2019, all ofwhich are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedRTS21920B_SequenceListing_ST25.txt created on 9 Dec. 2019 and having asize of 714,386 bytes and is filed concurrently with the specification.The sequence listing comprised in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD

The disclosure relates to the field of molecular biology, in particularto compositions of novel RNA-guided Cas endonuclease systems, andcompositions and methods for editing or modifying the genome of a cell.

BACKGROUND

Recombinant DNA technology has made it possible to insert DNA sequencesat targeted genomic locations and/or modify specific endogenouschromosomal sequences. Site-specific integration techniques, whichemploy site-specific recombination systems, as well as other types ofrecombination technologies, have been used to generate targetedinsertions of genes of interest in a variety of organism. Genome-editingtechniques such as designer zinc finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), or homing meganucleases, areavailable for producing targeted genome perturbations, but these systemstend to have low specificity and employ designed nucleases that need tobe redesigned for each target site, which renders them costly andtime-consuming to prepare.

Newer technologies utilizing archaeal or bacterial adaptive immunitysystems have been identified, called CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats), which comprise different domainsof effector proteins that encompass a variety of activities (DNArecognition, binding, and optionally cleavage).

Despite the identification and characterization of some of thesesystems, there remains a need for identifying novel effectors andsystems, as well as demonstrating activity in eukaryotes, particularlyanimals and plants, to effect editing of endogenous andpreviously-introduced heterologous polynucleotides.

Herein is described a novel Cas endonuclease, “Cas-alpha”, exemplaryproteins, and methods and compositions for use thereof.

SUMMARY

Disclosed herein are compositions of novel Cas endonucleases and methodsof use thereof. These endonucleases, of the novel class Cas-alpha, arecapable of being guided by a guide polynucleotide to target and cleavedouble-stranded DNA in a PAM-dependent fashion, as demonstrated inprokaryotes (E. coli) and three different kingdoms of eukaryotes: plant,animal, and fungi. The

In one aspect, a synthetic composition is provided, comprising aCRISPR-Cas endonuclease comprising at least one zinc-finger-like domain,at least one bridge-helix-like domain, a tri-split RuvC domain(comprising non-contiguous RuvC-I domain, RuvC-II domain, and RuvC-IIIdomain), optionally comprising a heterologous polynucleotide.

In any aspect, in any of the compositions or methods, at least onecomponent that has been optimized for expression in a eukaryotic cell,particularly a plant cell, a fungal cell, or an animal cell, isprovided.

In one aspect, a synthetic composition is provided, comprising apolynucleotide encoding CRISPR-Cas effector protein derived from anorganism selected from the group consisting of: Acidibacillussulfuroxidans, Alicyclobacillus acidoterrestris, Aneurinibacillusdanicus, Archaea, Bacillus, Bacillus cereus, Bacillus megaterium,Bacillus pseudomycoides, Bacillus sp., Bacillus thuringiensis, Bacillustoyonensis, Bacillus wiedmannii, Bacteroides plebeius, Bos taurus,Brevibacillus centrosporus, Candidatus Aureabacteria bacterium,Candidatus Levybacteria bacterium, Candidatus Micrarchaeota archaeon,Cellulosilyticum ruminicola, Clostridioides difficile, Clostridiumbotulinum, Clostridium fallax, Clostridium hiranonis, Clostridiumihumii, Clostridium novyi, Clostridium paraputrificum, Clostridiumpasteurianum, Clostridium perfringens, Clostridium sp., Clostridiumtetani, Clostridium ventriculi, Desulfovibrio fructosivorans, Dorealongicatena, Eubacterium siraeum, Flavobacterium thermophilum, Gallusgallus, Hepatitis delta virus, Homo sapiens, Human betaherpesvirus 5,Hydrogenivirga sp., Mus musculus, Parageobacillus thermoglucosidasius,Peptoclostridium sp., Phascolarctobacterium sp., Prevotella copri,Ruminiclostridium hungatei, Ruminococcus albus, Ruminococcus sp.,Saccharomyces cerevisiae, Simian virus 40, Solanum tuberosum,Sulfurihydrogenibium azorense, Syntrophomonas palmitatica, Tobacco etchvirus, and Zea mays; and a heterologous polynucleotide.

In one aspect, a synthetic composition is provided, comprising: aeukaryotic cell, a heterologous CRISPR-Cas effector; wherein saidheterologous CRISPR-Cas effector protein comprises fewer than 800,between 790 and 800, fewer than 790, between 780 and 790, fewer than780, between 780 and 770, fewer than 770, between 770 and 760, fewerthan 760, between 760 and 750, fewer than 750, between 750 and 740,fewer than 740, between 740 and 730, fewer than 730, between 730 and720, fewer than 720, between 720 and 710, fewer than 710, between 710and 700, or even fewer than 700 amino acids, such as fewer than 700,fewer than 790, fewer than 780, fewer than 750, fewer than 700, fewerthan 650, fewer than 600, fewer than 550, fewer than 500, fewer than450, fewer than 400, fewer than 350, or even fewer than 350 amino acids.

In one aspect, a synthetic composition is provided that comprises aCRISPR-Cas endonuclease, wherein said CRISPR-Cas endonuclease comprises,when aligned to SEQ ID NO:17, relative to the amino acid positionnumbers of SEQ ID NO:17, at least one, at least two, at least three, atleast four, at least five, at least six, or seven of the following: aGlycine (G) at position 337, a Glycine (G) at position 341, a GlutamicAcid (E) at position 430, a Leucine (L) at position 432, a Cysteine (C)at position 487, a Cysteine (C) at position 490, a Cysteine (C) atposition 507, and/or a Cysteine (C) or Histidine (H) at position 512.

In one aspect, a synthetic composition is provided that comprises aCRISPR-Cas endonuclease, wherein said CRISPR-Cas endonuclease comprisesone, two, or three of the following motifs: GxxxG, ExL, and/or one ormore Cx_(n)(C,H) (where n=one or more amino acids).

In one aspect, a synthetic composition is provided that comprises aCRISPR-Cas endonuclease, wherein said CRISPR-Cas endonuclease comprisesone or more zinc finger motifs.

In one aspect, a synthetic composition is provided comprising aCRISPR-Cas effector protein sharing at least 50%, between 50% and 55%,at least 55%, between 55% and 60%, at least 60%, between 60% and 65%, atleast 65%, between 65% and 70%, at least 70%, between 70% and 75%, atleast 75%, between 75% and 80%, at least 80%, between 80% and 85%, atleast 85%, between 85% and 90%, at least 90%, between 90% and 95%, atleast 95%, between 95% and 96%, at least 96%, between 96% and 97%, atleast 97%, between 97% and 98%, at least 98%, between 98% and 99%, atleast 99%, between 99% and 100%, or 100% sequence identity with at least250, between 250 and 300, at least 300, between 300 and 350, at least350, between 350 and 400, at least 400, or greater than 400 contiguousamino acids of a sequence selected from the group consisting of SEQ IDNOs:17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,and 371.

In one aspect, a synthetic composition is provided comprising apolynucleotide encoding a CRISPR-Cas effector protein sharing at least50%, between 50% and 55%, at least 55%, between 55% and 60%, at least60%, between 60% and 65%, at least 65%, between 65% and 70%, at least70%, between 70% and 75%, at least 75%, between 75% and 80%, at least80%, between 80% and 85%, at least 85%, between 85% and 90%, at least90%, between 90% and 95%, at least 95%, between 95% and 96%, at least96%, between 96% and 97%, at least 97%, between 97% and 98%, at least98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100%sequence identity with at least 250, between 250 and 500, at least 500,between 500 and 600, at least 600, between 600 and 700, at least 700,between 700 and 750, at least 750, between 750 and 800, at least 800,between 800 and 850, at least 850, between 850 and 900, at least 900,between 900 and 950, at least 950, between 950 and 1000, at least 1000,or greater than 1000 amino acids of a polypeptide selected from thegroup consisting of SEQ ID NOs:17, 18, 19, 20, 32, 33, 34, 35, 36, 37,38, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, and 371.

In one aspect, a synthetic composition is provided comprising apolynucleotide encoding a CRISPR-Cas effector protein that is capable ofhybridizing with a polynucleotide sharing at least 50%, between 50% and55%, at least 55%, between 55% and 60%, at least 60%, between 60% and65%, at least 65%, between 65% and 70%, at least 70%, between 70% and75%, at least 75%, between 75% and 80%, at least 80%, between 80% and85%, at least 85%, between 85% and 90%, at least 90%, between 90% and95%, at least 95%, between 95% and 96%, at least 96%, between 96% and97%, at least 97%, between 97% and 98%, at least 98%, between 98% and99%, at least 99%, between 99% and 100%, or 100% sequence identity withat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, 30, or greater than 30contiguous nucleotides of an RNA sequence selected from the groupconsisting of SEQ ID NOs:57, 58, 59, 64, 65, 66, 67, 68, 73, 74, 75, 76,77, 102, 103, 104, 105, 177, 178, 179, 180, 181, 182, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 230, 231, 232, 233, 234, 238, 240, 241, 245, 246,247, 248, 252, and 253.

Any of the methods or compositions herein may further comprise aheterologous polynucleotide. The heterologous polynucleotide may beselected from the group consisting of: a noncoding regulatory expressionelement such as a promoter, intron, enhancer, or terminator; a donorpolynucleotide; a polynucleotide modification template, optionallycomprising at least one nucleotide modification as compared to thesequence of a polynucleotide in a cell; a transgene; a guide RNA; aguide DNA; a guide RNA-DNA hybrid; an endonuclease; a nuclearlocalization signal; and a cell transit peptide.

In one aspect, methods are provided for using any of the compositionsdisclosed herein. In some embodiments, methods are provided for aCas-alpha endonuclease to bind to a target sequence of a polynucleotide,for example in the genome of a cell or in vitro. In some embodiments,the Cas-alpha endonuclease forms a complex with a guide polynucleotide,for example a guide RNA. In some embodiments, the complex recognizes,binds to, and optionally creates a nick (one strand) or a break (twostrands) in the polynucleotide at or near the target sequence. In someembodiments, the nick or break is repaired via Non-Homologous EndJoining (NHEJ). In some embodiments, the nick or break is repaired viaHomology-Directed Repair (HDR) or via Homologous Recombination (HR),with a polynucleotide modification template or a donor DNA molecule.

The novel Cas endonucleases described herein are capable of creating adouble-strand break in, or adjacent to, a target polynucleotide thatcomprises an appropriate PAM, and to which it is directed by a guidepolynucleotide, in any prokaryotic or eukaryotic cell. In some cases,the cell is a plant cell or an animal cell or a fungal cell. In somecases, a plant cell is selected from the group consisting of: maize,soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye,barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa,sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, andtomato.

BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIGS. 1A-1D depict the intact CRISPR-Cas system comprising all of thecomponents required for acquisition and interference. These includedgenes that together encoded all the proteins needed for acquiring andintegrated spacers (Cas1 and Cas2) and a novel protein comprising a DNAcleavage domain, Cas-alpha (a), in an operon-like structure adjacent toa CRISPR array. Additionally, a gene encoding a protein with homology toCas4 was also encoded in the locus. FIG. 1A depicts the locusarchitecture for the Cas-alpha 1, Cas-alpha 3, and Cas-alpha 4 systems.FIG. 1B depicts the locus architecture for the Cas-alpha2 system. FIG.1C depicts the locus architecture for the Cas-alpha 6 system. FIG. 1Ddepicts the locus architecture for the Cas-alpha 5, 7, 8, 9, 10, and 11systems.

FIG. 2 shows a detailed structural examination of the Cas-alpha protein,depicting distinct differences from previously described Class 2endonucleases. Conserved residues are indicated. Key residues involvedin DNA cleavage are indicated with an asterisk. Numbers correspond tothe Cas-alpha 1 protein.

FIG. 3 outlines a method of detection of double stranded DNA targetrecognition and cleavage, using cell lysates expressing a Cas-alphaendonuclease.

FIGS. 4A-4E show cleavage of a target polynucleotide by the Cas-alpha 1endonuclease at nucleotide position 21. FIG. 4A shows data for theCas-alpha 1 negative control, FIG. 4B shows data for Cas-alpha 1 usingthe entire (complete) CRISPR locus whose CRISPR array was modified todirect cleavage at the target polynucleotide, FIG. 4C shows the data forthe Cas-alpha 1 complete locus plus when expression was enhanced using aT7 promoter, FIG. 4D shows the data for the Cas-alpha 1 minimal locuswhen expression was enhanced using a T7 promoter, and FIG. 4E shows thedata for the reaction without Cas-alpha 1 but with the rest of theCRISPR locus when expression was enhanced with a T7 promoter.

FIGS. 5A-5B depict schematics to determine the orientation of PAMrecognition relative to spacer recognition, guide RNA(s) were designedto base pair with either the sense or anti-sense strands of the T2target. If the guide RNA(s) designed to base pair with the sense strandresult in the recovery of PAM preferences and yield a cleavage signal,then the protospacer is on the anti-sense strand and PAM recognitionoccurs 3′ relative to it (FIG. 5A). Conversely, if the guide RNA(s)designed to base pair with the anti-sense strand produce PAM preferencesand a cleavage signal, then the protospacer is on the sense strand andPAM recognition occurs in an orientation 5′ to it (FIG. 5B).

FIGS. 6A-6E show cleavage of a target polynucleotide by the Cas-alpha 4endonuclease at nucleotide position 24. FIG. 6A shows the data for theCas-alpha 4 negative control. FIG. 6B shows the data for Cas-alpha 4plus the T2-1 sgRNA. FIG. 6C shows the data for Cas-alpha 4 plus theT2-2 sgRNA. FIG. 6D shows the data for Cas-alpha 4 plus T2-1crRNA/tracrRNA. FIG. 6E shows the data for the Cas-alpha 4 plus T2-2crRNA/tracrRNA.

FIGS. 7A-7K show representative Cas-alpha loci, endonucleases, proteins,guide RNA components, and other sequences that have been identified froma variety of bacterial and archaebacterial organisms, including:Candidatus Micrarchaeota archaeon (FIGS. 7A, 7B, 7E), CandidatusAureabacteria bacterium (FIG. 7C), various uncultured bacteria (FIGS.7D, 7F), Parageobacillus thermoglucosidasius (FIG. 7G), Acidibacillussulfuroxidans (FIG. 7H), Ruminococcus sp. (FIG. 7I), Syntrophomonaspalmitatica (FIG. 7J), and Clostridium novyi (FIG. 7K).

FIGS. 8A-8K shows distinct structural features in the representativeCas-alpha proteins, with the protein sequence in bold text. The non-boldcharacters beneath each amino acid residue indicate the likely secondarystructure feature, with C representing a non-structured element or coil,E representing a beta strand, and H representing an alpha helix. Zincfinger domains are depicted by dashed line boxes, and stars denote keyamino acid residues involved in zinc ion binding. RuvC subdomains of thesplit RuvC domains are depicted by solid line boxes. A bridge helix isdepicted by dotted-dashed line boxes. Coiled coils are depicted by solidline cylinders. Solid plus signs denote key catalytic residuescharacteristic of RuvC domain motifs. FIG. 8A depicts Cas-alpha 1 fromCandidatus Micrarchaeota archaeon (SEQ ID NO:17), FIG. 8B depictsCas-alpha 2 from Candidatus Micrarchaeota archaeon (SEQ ID NO:18), FIG.8C depicts Cas-alpha 3 from Candidatus Aureabacteria bacterium (SEQ IDNO:19), FIG. 8D depicts Cas-alpha 4 from an uncultured bacterium (SEQ IDNO:20), FIG. 8E depicts Cas-alpha 5 from Candidatus Micrarchaeotaarchaeon (SEQ ID NO:32), FIG. 8F depicts Cas-alpha 6 from an unculturedbacterium (SEQ ID NO:33), FIG. 8G depicts Cas-alpha 7 fromParageobacillus thermoglucosidasius (SEQ ID NO:34), FIG. 811 depictsCas-alpha 8 from Acidibacillus sulfuroxidans (SEQ ID NO:35), FIG. 81depicts Cas-alpha 9 from Ruminococus sp. (SEQ ID NO:36), FIG. 8J depictsCas-alpha1 0 from Syntrophomonas palmitatica (SEQ ID NO:37) whichfeatures a unique motif of three zinc finger domains, FIG. 8K depictsCas-alpha 11 from Clostridium novyi (SEQ ID NO:38). Whole genomesequencing of the organism comprising Cas-alpha 11 showed that theCas-alpha locus was the only CRISPR system in that organism.

FIG. 9A depicts how the Cas-alpha protein subunits interact with thehybrid duplex of the target DNA and the guide RNA. FIG. 9B is athree-dimensional model of the C-terminal half of Cas-alpha 4, showingthe regions identified as the Helical Hairpin/Bridge Helix region commonto Cas proteins, the RuvC domain, and the Zinc Finger motif.

FIGS. 10A-10D depict example expression constructs for use of aCas-alpha endonuclease in a eukaryotic cell. FIG. 10A is an example of ahuman cell Cas-alpha DNA expression construct. FIG. 10B is an example ofa plant cell Cas-alpha DNA expression construct. FIG. 10C is an exampleof a yeast (Saccharomyces cerevisiae) Cas-alpha DNA expressionconstruct. FIG. 10D is an example of an inducible yeast (Saccharomycescerevisiae) Cas-alpha DNA expression construct.

FIGS. 11A-11D depicts examples of eukaryotic optimized Cas-alpha guideRNA expression constructs. FIG. 11A is an example of a human cell singleguide RNA (sgRNA) DNA expression construct. FIG. 11B is an example of aplant cell single guide RNA (sgRNA) DNA expression construct. FIG. 11Cis an example of a yeast (Saccharomyces cerevisiae) single guide RNA(sgRNA) DNA expression construct. FIG. 11D is another example of a plantcell single guide RNA (sgRNA) DNA expression construct.

FIG. 12 depicts examples of engineered genes for recombinant expressionand purification of Cas-alpha endonucleases in E. coli.

FIG. 13 shows double stranded break repair mutations in plant cells fromCas-alpha endonuclease activity. Depicted are mutations resulting fromCas-alpha 4 in Zea mays. The WT Reference is SEQ ID NO:120, Mutation 1is SEQ ID NO: 121, Mutation 2 is SEQ ID NO:122, Mutation 3 is SEQ IDNO:123, and Mutation 4 is SEQ ID NO:124.

FIGS. 14A-14B show double stranded break repair mutations in animalcells from Cas-alpha endonuclease activity. FIG. 14A depicts indelmutations (VEGFA Target 2 Mutations 1-5 given as SEQ ID NOs: 127-131,compared to the WT Reference SEQ ID NO:126; VEGFA Target 3 (Mutationgiven as SEQ ID NO:133, compared to the WT reference SEQ ID NO:132)resulting from Cas-alpha4 RNP electroporation. FIG. 14B depicts indelmutations VEGFA Target 3 (Mutations 1 and 2 given as SEQ ID NO:134-135,compared to the WT reference SEQ ID NO:132) resulting from Cas-alpha4and sgRNA DNA expression cassette lipofection.

FIGS. 15A-15D show Cas-alpha4 double-stranded DNA target cleavage. FIG.15A shows that a supercoiled (SC) plasmid DNA containing a guide RNAtarget (˜20 bp) immediately 3′ of a PAM (5′-TTTR-3′ where R representedeither A or G bps) was completely converted to a linear form (FLL),thus, illustrating the formation of a dsDNA break. Additionally,cleavage of linear DNA resulted in DNA fragments of an expected sizefurther validating Cas-alpha 4 mediated dsDNA break formation. FIG. 15Bshows that Cas-alpha 4 requires a PAM and guide RNA to cleave a dsDNAtarget. FIG. 15C shows that Cas-alpha 4 generates 5′ staggeredoverhanging DNA cut-sites, with cleavage predominantly occurringcentered around positions 20-24 bp in respect to the PAMsequence. FIG.15D shows trans-acting ssDNase activity of Cas-alpha 4 that wasactivated by dsDNA only in the presence of a guide RNA.

FIGS. 16A-16T show double stranded DNA target cleavage activity for allCas-alpha endonucleases except Cas-alpha 5. FIG. 16A is a negativecontrol (−IPTG). FIG. 16B is a negative control (+IPTG). FIG. 16C showscleavage of a double stranded DNA target by Cas-alpha 2 (−IPTG) atprotospacer position 21. FIG. 16D shows cleavage of a double-strandedDNA target by Cas-alpha2 (+IPTG) at protospacer position 21. FIG. 16Eshows no cleavage of a double stranded DNA target by Cas-alpha 3(−IPTG). FIG. 16F shows cleavage of a double stranded DNA target byCas-alpha 3 (+IPTG) at protospacer position 21. FIG. 16G shows nocleavage of a double stranded DNA target by Cas-alpha 5 (−IPTG). FIG.16H shows no cleavage of a double stranded DNA target by Cas-alpha 5(−IPTG). FIG. 16I shows cleavage of a double stranded DNA target byCas-alpha 6 (−IPTG). FIG. 16J shows no cleavage of a double stranded DNAtarget by Cas-alpha 6 (+IPTG) at protospacer position 24. FIG. 16K showscleavage of a double stranded DNA target by Cas-alpha 7 (−IPTG) atprotospacer position 24. FIG. 16L shows cleavage of a double strandedDNA target by Cas-alpha 7 (+IPTG) at protospacer position 24. FIG. 16Mshows no cleavage of a double-stranded DNA target by Cas-alpha8 (−IPTG).FIG. 16N shows cleavage of a double stranded DNA target by Cas-alpha 8(+IPTG) at protospacer position 24. FIG. 16O shows cleavage of a doublestranded DNA target by Cas-alpha 9 (−IPTG) at protospacer position 24.FIG. 16P shows cleavage of a double stranded DNA target by Cas-alpha9(+IPTG) at protospacer position 24. FIG. 16Q shows cleavage of a doublestranded DNA target by Cas-alpha 10 (−IPTG) at protospacer position 24.FIG. 16R shows cleavage of a double stranded DNA target by Cas-alpha 10(+IPTG) at protospacer position 24. FIG. 16S shows cleavage of a doublestranded DNA target by Cas-alpha 11 (−IPTG) at protospacer position 24.FIG. 16T shows cleavage of a double stranded DNA target by Cas-alpha 11(+IPTG) at protospacer position 24.

FIG. 17A depicts one method to assess Cas-alpha double stranded DNAtarget cleavage in E. coli cells. FIGS. 17B-17E show double stranded DNAtarget cleavage in E. coli. The “no target” experiments provide abaseline for transformation efficiency in the absence of double strandedDNA target cleavage. “Target” experiments, PAM+T2, were performed withand without IPTG (0.5 mM) to examine target cleavage under differentCas-alpha endonuclease and guide RNA expression conditions. FIG. 17Bshows results for Cas-alpha 2 and Cas-alpha 3. FIG. 17C shows resultsfor Cas-alpha 6 and Cas-alpha 7. FIG. 17D shows results for Cas-alpha 8and Cas-alpha 9. FIG. 17E shows results for Cas-alpha 10 and Cas-alpha11.

FIGS. 18A-18B depict double stranded break repair mutations in plantcells from Cas-alpha endonuclease activity, for particle gun experimentsdelivering Cas-alpha 10 DNA expression constructions into Zea maysimmature embryos. FIG. 18A shows recovery of targeted deletions producedat or near the nuclease cut site for the nptII target site. FIG. 18Bshows recovery of targeted deletions produced at or near the nucleasecut site for the ms26 target site.

FIG. 19A depicts the experimental design for homology-directed repair ina eukaryotic cell, Saccaromyces cerevisiae. An exogenously supplied DNArepair template (double stranded) with homology flanking a Cas-alpha 10target site was used to introduce one or two premature stop codons(depending on the DNA repair outcome) in the ade2 gene following aCas-alpha 10 induced double strand break (DSB). To avoid targeting ofthe repair template, it also contained a T to A change in the PAM regionfor Cas-alpha 10. FIG. 19B shows that a red cellular phenotypeindicative of ade2 gene disruption was recovered when both the repairtemplate and Cas-alpha 10 and sgRNA expression constructs weretransformed, and a double strand break was created by the Cas-alphaendonuclease and repaired with a template (HDR). FIG. 19C showssequencing results of the Cas-alpha10 ade2 gene target site, confirmingthe introduction of at least one stop codon in 3 independent redcolonies (labeled “1”, “2” and “3”). Stop codons were introduced intothe antisense frame. SEQ ID NO:170 The reference DNA sequence fromSaccharomyces cerevisiae is given as SEQ ID NO:170, the repair templateDNA is SEQ ID NO:171, Red Colony 1 repair outcome 1 is SEQ ID NO:172,Red Colony 1 repair outcome 2 is SEQ ID NO:173, Red Colony 2 repairoutcome 1 is SEQ ID NO:174, Red Colony 3 repair outcome 1 is SEQ ID NO:175, and Red Colony 3 repair outcome 2 is SEQ ID NO:176.

FIG. 20 shows the phylogenetic relationships among some of the Cas-alphaorthologs. Three supergroups were identified (I, II, and III). Group Icomprised Clade 1 (Candidate Archaea and Aureabacteria (Cas1, Cas2, Cas4typically encoded in the locus)). GroupII comprised Clade 2 (Aquificae(Sulfurihydrogenibium and Hydrogenivirga genera) and Deltaproteobacteria(Desulfovibrio genus)), Clade 3 (Candidate Archaea (Cas1, Cas2, and Cas4typically encoded in the locus)), Clade 4 (Bacteroidetes (Prevotella andBacteroides genera)), Clade 5 (Candidate Levybacterium), and Clade 6(Clostridia (Dorea, Ruminococcus, Clostridium, Clostridioides,Peptocolstridium, Cellulosilyticym, Eubacterium, Syntrophomonasgenera)). Group III comprised Clade 7 (Bacilli (Bacillus, Acidibacillus,Aneurinibacillus, Brevibacillus, Parageobacillus, Alicyclobacillusgenera)), Clade 8 (Negativicutes (Phascolarctobacterium genus)), andClade 9 (Flavobacteriia (Flavobacterium genus)). A diamond symbolrepresents the Cas-alphas 1-11 endonucleases described herein.

FIG. 21A illustrates a transposase (Tnp) associated Cas-alpha CRISPRsystem. In both instances, a Tnp-like protein is encoded upstream of aCas-alpha endonuclease and CRISPR array. FIG. 21B shows a Cas-alphaendonuclease and guide RNA in complex with its target site and aTnp-like protein that is posed to integrate a DNA Payload(circle with adashed-line) within or near the Cas-alpha double stranded DNA targetsite.

The sequence descriptions and sequence listing attached hereto complywith the rules governing nucleotide and amino acid sequence disclosuresin patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825. Thesequence descriptions comprise the three letter codes for amino acids asdefined in 37 C.F.R. §§ 1.821 and 1.825, which are incorporated hereinby reference.

-   -   SEQ ID NO:1 is the Cas1 encoded in Cas-alpha 1 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:2 is the Cas1 encoded in Cas-alpha 2 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:3 is the Cas1 encoded in Cas-alpha 3 locus PRT        sequence from Candidatus Aureabacteria bacterium.    -   SEQ ID NO:4 is the Cas1 encoded in Cas-alpha 4 locus PRT        sequence from Uncultured archaeon.    -   SEQ ID NO:5 is the Cas2 encoded in Cas-alpha 1 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:6 is the Cas2 encoded in Cas-alpha 2 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:7 is the Cas2 encoded in Cas-alpha 3 locus PRT        sequence from Candidatus Aureabacteria bacterium.    -   SEQ ID NO:8 is the Cas2 encoded in Cas-alpha 4 locus PRT        sequence from Uncultured archaeon.    -   SEQ ID NO:9 is the Cas4 encoded in Cas-alpha 1 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:10 is the Cas4 encoded in Cas-alpha 2 locus PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:11 is the Cas4 encoded in Cas-alpha 3 locus PRT        sequence from Candidatus Aureabacteria bacterium.    -   SEQ ID NO:12 is the Cas4 encoded in Cas-alpha 4 locus PRT        sequence from Uncultured archaeon.    -   SEQ ID NO:13 is the Cas-alpha 1 endonuclease gene DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:14 is the Cas-alpha 2 endonuclease gene DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:15 is the Cas-alpha 3 endonuclease gene DNA sequence        from Candidatus Aureabacteria bacterium.    -   SEQ ID NO:16 is the Cas-alpha 4 endonuclease gene DNA sequence        from Uncultured archaeon.    -   SEQ ID NO:17 is the Cas-alpha 1 endonuclease (Cas14b4) PRT        sequence from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:18 is the Cas-alpha 2 endonuclease PRT sequence from        Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:19 is the Cas-alpha 3 endonuclease PRT sequence from        Candidatus Aureabacteria bacterium.    -   SEQ ID NO:20 is the Cas-alpha 4 endonuclease (Cas14a1) PRT        sequence from Uncultured archaeon.    -   SEQ ID NO:21 is the Cas-alpha 1 locus DNA sequence from        Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:22 is the Cas-alpha 2 locus DNA sequence from        Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:23 is the Cas-alpha 3 locus DNA sequence from        Candidatus Aureabacteria bacterium.    -   SEQ ID NO:24 is the Cas-alpha 4 locus DNA sequence from        Uncultured archaeon.    -   SEQ ID NO:25 is the Cas-alpha 5 endonuclease gene DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:26 is the Cas-alpha 6 endonuclease gene DNA sequence        from Uncultured archaeon.    -   SEQ ID NO:27 is the Cas-alpha 7 endonuclease gene DNA sequence        from Parageobacillus thermoglucosidasius.    -   SEQ ID NO:28 is the Cas-alpha 8 endonuclease gene DNA sequence        from Acidibacillus sulfuroxidans.    -   SEQ ID NO:29 is the Cas-alpha 9 endonuclease gene DNA sequence        from Ruminococcus sp.    -   SEQ ID NO:30 is the Cas-alpha 10 endonuclease gene DNA sequence        from Syntrophomonas palmitatica.    -   SEQ ID NO:31 is the Cas-alpha 11 endonuclease gene DNA sequence        from Clostridium novyi.    -   SEQ ID NO:32 is the Cas-alpha 5 endonuclease PRT sequence from        Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:33 is the Cas-alpha 6 endonuclease PRT sequence from        Uncultured archaeon.    -   SEQ ID NO:34 is the Cas-alpha 7 endonuclease PRT sequence from        Parageobacillus thermoglucosidasius.    -   SEQ ID NO:35 is the Cas-alpha 8 endonuclease PRT sequence from        Acidibacillus sulfuroxidans.    -   SEQ ID NO:36 is the Cas-alpha 9 endonuclease PRT sequence from        Ruminococcus sp.    -   SEQ ID NO:37 is the Cas-alpha 10 endonuclease PRT sequence from        Syntrophomonas palmitatica.    -   SEQ ID NO:38 is the Cas-alpha 11 endonuclease PRT sequence from        Clostridium novyi.    -   SEQ ID NO:39 is the Cas-alpha 5 locus DNA sequence from        Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:40 is the Cas-alpha 6 locus DNA sequence from        Uncultured archaeon.    -   SEQ ID NO:41 is the Cas-alpha 7 locus DNA sequence from        Parageobacillus thermoglucosidasius.    -   SEQ ID NO:42 is the Cas-alpha 8 locus DNA sequence from        Acidibacillus sulfuroxidans.    -   SEQ ID NO:43 is the Cas-alpha 9 locus DNA sequence from        Ruminococcus sp.    -   SEQ ID NO:44 is the Cas-alpha 10 locus DNA sequence from        Syntrophomonas palmitatica.    -   SEQ ID NO:45 is the Cas-alpha 11 locus DNA sequence from        Clostridium novyi.    -   SEQ ID NO:46 is the Cas-alpha 1 repeat consensus DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:47 is the Cas-alpha 2 repeat consensus DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:48 is the Cas-alpha 3 repeat consensus DNA sequence        from Candidatus Aureabacteria bacterium.    -   SEQ ID NO:49 is the Cas-alpha 4 repeat consensus DNA sequence        from Uncultured archaeon.    -   SEQ ID NO:50 is the Cas-alpha 5 repeat consensus DNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:51 is the Cas-alpha 6 repeat consensus DNA sequence        from Uncultured archaeon.    -   SEQ ID NO:52 is the Cas-alpha 7 repeat consensus DNA sequence        from Parageobacillus thermoglucosidasius.    -   SEQ ID NO:53 is the Cas-alpha 8 repeat consensus DNA sequence        from Acidibacillus sulfuroxidans.    -   SEQ ID NO:54 is the Cas-alpha 9 repeat consensus DNA sequence        from Ruminococcus sp.    -   SEQ ID NO:55 is the Cas-alpha 10 repeat consensus DNA sequence        from Syntrophomonas palmitatica.    -   SEQ ID NO:56 is the Cas-alpha 11 repeat consensus DNA sequence        from Clostridium novyi.    -   SEQ ID NO:57 is the Cas-alpha 1 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:58 is the Cas-alpha 2 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:59 is the Cas-alpha 4 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:60 is the Cas-alpha 1 tracrRNA version 1 RNA sequence        from Candidatus Micrarchaeota archaeon.

SEQ ID NO:61 is the Cas-alpha 1 tracrRNA version 2 RNA sequence fromCandidatus Micrarchaeota archaeon.

-   -   SEQ ID NO:62 is the Cas-alpha 1 tracrRNA version 3 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:63 is the Cas-alpha 1 tracrRNA version 4 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:64 is the Cas-alpha 2 tracrRNA version 1 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:65 is the Cas-alpha 2 tracrRNA version 2 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:66 is the Cas-alpha 2 tracrRNA version 3 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:67 is the Cas-alpha 2 tracrRNA version 4 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:68 is the Cas-alpha 4 tracrRNA version 1 RNA sequence        from Uncultured archaeon.    -   SEQ ID NO:69 is the Cas-alpha 1 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:70 is the Cas-alpha 1 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:71 is the Cas-alpha 1 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:72 is the Cas-alpha 1 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:73 is the Cas-alpha 2 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:74 is the Cas-alpha 2 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:75 is the Cas-alpha 2 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:76 is the Cas-alpha 2 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:77 is the Cas-alpha 4 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:78 is the T2 spacer DNA sequence from Artificial.    -   SEQ ID NO:79 is the Complete Cas-alpha 1 locus engineered to        target T2 DNA sequence from Artificial.    -   SEQ ID NO:80 is the Minimal Cas-alpha 1 locus engineered to        target T2 DNA sequence from Artificial.    -   SEQ ID NO:81 is the 10× histidine tag PRT sequence from        Artificial.    -   SEQ ID NO:82 is the 6× histidine tag PRT sequence from        Artificial.    -   SEQ ID NO:83 is the Maltose binding protein tag PRT sequence        from Artificial.    -   SEQ ID NO:84 is the Tobacco etch virus cleavage site PRT        sequence from Tobacco etch virus.    -   SEQ ID NO:85 is the A1 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:86 is the A2 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:87 is the R0 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:88 is the C0 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:89 is the F1 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:90 is the R1 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:91 is the Bridge amplification portion of F1        oligonucleotide DNA sequence from Artificial.    -   SEQ ID NO:92 is the Bridge amplification portion of R1        oligonucleotide DNA sequence from Artificial.    -   SEQ ID NO:93 is the F2 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:94 is the R2 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:95 is the C1 oligonucleotide DNA sequence from        Artificial.    -   SEQ ID NO:96 is the Sequence resulting from cleavage and adapter        ligation at position 21 of the target DNA sequence from        Artificial.    -   SEQ ID NO:97 is the Adapter portion of SEQ ID NO. 96 DNA        sequence from Artificial.    -   SEQ ID NO:98 is the Target portion of SEQ ID NO. 96 DNA sequence        from Artificial.    -   SEQ ID NO:99 is the Sequence 5′ of PAM DNA sequence from        Artificial.    -   SEQ ID NO:100 is the Fixed double stranded DNA target DNA        sequence from Artificial.    -   SEQ ID NO:101 is the T2 target sequence DNA sequence from        Artificial.    -   SEQ ID NO:102 is the Cas-alpha 4 T2-1 sgRNA RNA sequence from        Artificial.    -   SEQ ID NO:103 is the Cas-alpha 4 T2-2 sgRNA RNA sequence from        Artificial.    -   SEQ ID NO:104 is the Cas-alpha 4 T2-1 crRNA RNA sequence from        Artificial.    -   SEQ ID NO:105 is the Cas-alpha 4 T2-2 crRNA RNA sequence from        Artificial.    -   SEQ ID NO:106 is the ST-LS1 intron 2 DNA sequence from Solanum        tuberosum.    -   SEQ ID NO:107 is the SV40 NLS PRT sequence from Simian virus 40.    -   SEQ ID NO:108 is the Nuc NLS PRT sequence from Mus musculus.    -   SEQ ID NO:109 is the Maize UBI promoter DNA sequence from Zea        mays.    -   SEQ ID NO:110 is the Chicken beta-actin promoter DNA sequence        from Gallus gallus.    -   SEQ ID NO:111 is the CMV enhancer DNA sequence from Human        beta-herpesvirus 5.    -   SEQ ID NO:112 is the Maize UBI 5 prime untranslated region DNA        sequence from Zea mays.    -   SEQ ID NO:113 is the Maize UBI intron 1 DNA sequence from Zea        mays.    -   SEQ ID NO:114 is the Hybrid intron DNA sequence from Artificial.    -   SEQ ID NO:115 is the Maize U6 polymerase III promoter DNA        sequence from Zea mays.    -   SEQ ID NO:116 is the Human U6 polymerase III promoter DNA        sequence from Homo sapiens.    -   SEQ ID NO:117 is the Strep II tag PRT sequence from Artificial.    -   SEQ ID NO:118 is the bGH poly(A) terminator DNA sequence from        Bos taurus.    -   SEQ ID NO:119 is the Potato Proteinase Inhibitor II (Pin II)        terminator DNA sequence from Solanum tuberosum.    -   SEQ ID NO:120 is the Zea mays Wt Reference (Liguleless Targets 2        and 3) DNA sequence from Zea mays.    -   SEQ ID NO:121 is the Mutation 1 (Liguleless Targets 2 and 3-DNA        Exp.) DNA sequence from Zea mays.    -   SEQ ID NO:122 is the Mutation 2 (Liguleless Targets 2 and 3-DNA        Exp.) DNA sequence from Zea mays.    -   SEQ ID NO:123 is the Mutation 3 (Liguleless Targets 2 and 3-DNA        Exp.) DNA sequence from Zea mays.    -   SEQ ID NO:124 is the Mutation 4 (Liguleless Targets 2 and 3-DNA        Exp.) DNA sequence from Zea mays.    -   SEQ ID NO:125 is the Mutation 5 (Liguleless Targets 2 and 3-DNA        Exp.) DNA sequence from Zea mays.    -   SEQ ID NO:126 is the HEK293 Wt Reference (VEGFA Target 2) DNA        sequence from Homo sapiens.    -   SEQ ID NO:127 is the Mutation 1 (VEGFA Target 2-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:128 is the Mutation 2 (VEGFA Target 2-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:129 is the Mutation 3 (VEGFA Target 2-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:130 is the Mutation 4 (VEGFA Target 2-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:131 is the Mutation 5 (VEGFA Target 2-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:132 is the HEK293 Wt Reference (VEGFA Target 3) DNA        sequence from Homo sapiens.    -   SEQ ID NO:133 is the Mutation 1 (VEGFA Target 3-RNP) DNA        sequence from Homo sapiens.    -   SEQ ID NO:134 is the Mutation 1 (VEGFA Target 3-DNA Exp) DNA        sequence from Homo sapiens.    -   SEQ ID NO:135 is the Mutation 2 (VEGFA Target 3-DNA Exp) DNA        sequence from Homo sapiens.    -   SEQ ID NO:136 is the ROX3 promoter DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:137 is the GAL promoter DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:138 is the HH Ribozyme (where N represents nucleotides        that are complementary to the 6 nucleotides 3′ of ribozyme) DNA        sequence from Artificial.    -   SEQ ID NO:139 is the HDV Ribozyme DNA sequence from Hepatitis        delta virus.    -   SEQ ID NO:140 is the SNR52 promoter DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:141 is the SUP4 terminator DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:142 is the FIG. 15C top sequence DNA sequence from        Artificial.    -   SEQ ID NO:143 is the FIG. 15C bottom sequence DNA sequence from        Artificial.    -   SEQ ID NO:144 is the FIG. 18A Reference DNA sequence from Zea        mays.    -   SEQ ID NO:145 is the Mutation 1 DNA sequence from Zea mays.    -   SEQ ID NO:146 is the Mutation 2 DNA sequence from Zea mays.    -   SEQ ID NO:147 is the Mutation 3 DNA sequence from Zea mays.    -   SEQ ID NO:148 is the Mutation 4 DNA sequence from Zea mays.    -   SEQ ID NO:149 is the Mutation 5 DNA sequence from Zea mays.    -   SEQ ID NO:150 is the Mutation 6 DNA sequence from Zea mays.    -   SEQ ID NO:151 is the Mutation 7 DNA sequence from Zea mays.    -   SEQ ID NO:152 is the Mutation 8 DNA sequence from Zea mays.    -   SEQ ID NO:153 is the Mutation 9 DNA sequence from Zea mays.    -   SEQ ID NO:154 is the Mutation 10 DNA sequence from Zea mays.    -   SEQ ID NO:155 is the Mutation 11 DNA sequence from Zea mays.    -   SEQ ID NO:156 is the Mutation 12 DNA sequence from Zea mays.    -   SEQ ID NO:157 is the Mutation 13 DNA sequence from Zea mays.    -   SEQ ID NO:158 is the Mutation 14 DNA sequence from Zea mays.    -   SEQ ID NO:159 is the Mutation 15 DNA sequence from Zea mays.    -   SEQ ID NO:160 is the Mutation 16 DNA sequence from Zea mays.    -   SEQ ID NO:161 is the Mutation 17 DNA sequence from Zea mays.    -   SEQ ID NO:162 is the Mutation 18 DNA sequence from Zea mays.    -   SEQ ID NO:163 is the Mutation 19 DNA sequence from Zea mays.    -   SEQ ID NO:164 is the FIG. 18B Reference DNA sequence from Zea        mays.    -   SEQ ID NO:165 is the Mutation 1 DNA sequence from Zea mays.    -   SEQ ID NO:166 is the Mutation 2 DNA sequence from Zea mays.    -   SEQ ID NO:167 is the Mutation 3 DNA sequence from Zea mays.    -   SEQ ID NO:168 is the Mutation 4 DNA sequence from Zea mays.    -   SEQ ID NO:169 is the Mutation 5 DNA sequence from Zea mays.    -   SEQ ID NO:170 is the FIG. 19C Reference DNA sequence from        Saccharomyces    -   cerevisiae.    -   SEQ ID NO:171 is the Repair template DNA sequence from        Artificial.    -   SEQ ID NO:172 is the Repair outcome 1 DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:173 is the Repair outcome 2 DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:174 is the Repair outcome 1 DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:175 is the Repair outcome 1 DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:176 is the Repair outcome 2 DNA sequence from        Saccharomyces cerevisiae.    -   SEQ ID NO:177 is the Cas-alpha 3 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:178 is the Cas-alpha 5 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:179 is the Cas-alpha 6 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:180 is the Cas-alpha 7 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:181 is the Cas-alpha 8 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:182 is the Cas-alpha 9 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:183 is the Cas-alpha 10 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:184 is the Cas-alpha 11 crRNA (where N represents any        nucleotide) RNA sequence from Artificial.    -   SEQ ID NO:185 is the Cas-alpha 2 tracrRNA version 5 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:186 is the Cas-alpha 2 tracrRNA version 6 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:187 is the Cas-alpha 2 tracrRNA version 7 RNA sequence        from Candidatus Micrarchaeota archaeon.    -   SEQ ID NO:188 is the Cas-alpha 6 tracrRNA version 1 RNA sequence        from Uncultured archaeon.    -   SEQ ID NO:189 is the Cas-alpha 6 tracrRNA version 2 RNA sequence        from Uncultured archaeon.    -   SEQ ID NO:190 is the Cas-alpha 6 tracrRNA version 3 RNA sequence        from Uncultured archaeon.    -   SEQ ID NO:191 is the Cas-alpha 6 tracrRNA version 4 RNA sequence        from Uncultured archaeon.    -   SEQ ID NO:192 is the Cas-alpha 7 tracrRNA version 1 RNA sequence        from Parageobacillus thermoglucosidasius.    -   SEQ ID NO:193 is the Cas-alpha 7 tracrRNA version 2 RNA sequence        from Parageobacillus thermoglucosidasius.    -   SEQ ID NO:194 is the Cas-alpha 8 tracrRNA version 1 RNA sequence        from Acidibacillus sulfuroxidans.    -   SEQ ID NO:195 is the Cas-alpha 8 tracrRNA version 2 RNA sequence        from Acidibacillus sulfuroxidans.    -   SEQ ID NO:196 is the Cas-alpha 8 tracrRNA version 3 RNA sequence        from Acidibacillus sulfuroxidans.    -   SEQ ID NO:197 is the Cas-alpha 9 tracrRNA version 1 RNA sequence        from Ruminococcus sp.    -   SEQ ID NO:198 is the Cas-alpha 9 tracrRNA version 2 RNA sequence        from Ruminococcus sp.    -   SEQ ID NO:199 is the Cas-alpha 10 tracrRNA version 1 RNA        sequence from Syntrophomonas palmitatica.    -   SEQ ID NO:200 is the Cas-alpha 10 tracrRNA version 2 RNA        sequence from Syntrophomonas palmitatica.    -   SEQ ID NO:201 is the Cas-alpha 10 tracrRNA version 3 RNA        sequence from Syntrophomonas palmitatica.    -   SEQ ID NO:202 is the Cas-alpha 10 tracrRNA version 4 RNA        sequence from Syntrophomonas palmitatica.    -   SEQ ID NO:203 is the Cas-alpha 10 tracrRNA version 5 RNA        sequence from Syntrophomonas palmitatica.    -   SEQ ID NO:204 is the Cas-alpha 11 tracrRNA version 1 RNA        sequence from Clostridium novyi.    -   SEQ ID NO:205 is the Cas-alpha 11 tracrRNA version 2 RNA        sequence from Clostridium novyi.    -   SEQ ID NO:206 is the Cas-alpha 11 tracrRNA version 3 RNA        sequence from Clostridium novyi.    -   SEQ ID NO:207 is the Cas-alpha 11 tracrRNA version 4 RNA        sequence from Clostridium novyi.    -   SEQ ID NO:208 is the Cas-alpha 2 sgRNA version 5 RNA sequence        from Artificial.    -   SEQ ID NO:209 is the Cas-alpha 2 sgRNA version 6 RNA sequence        from Artificial.    -   SEQ ID NO:210 is the Cas-alpha 2 sgRNA version 7 RNA sequence        from Artificial.    -   SEQ ID NO:211 is the Cas-alpha 6 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:212 is the Cas-alpha 6 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:213 is the Cas-alpha 6 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:214 is the Cas-alpha 6 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:215 is the Cas-alpha 7 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:216 is the Cas-alpha 7 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:217 is the Cas-alpha 7 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:218 is the Cas-alpha 8 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:219 is the Cas-alpha 8 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:220 is the Cas-alpha 8 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:221 is the Cas-alpha 8 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:222 is the Cas-alpha 9 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:223 is the Cas-alpha 9 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:224 is the Cas-alpha 9 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:225 is the Cas-alpha 10 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:226 is the Cas-alpha 10 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:227 is the Cas-alpha 10 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:228 is the Cas-alpha 10 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:229 is the Cas-alpha 10 sgRNA version 5 RNA sequence        from Artificial.    -   SEQ ID NO:230 is the Cas-alpha 11 sgRNA version 1 RNA sequence        from Artificial.    -   SEQ ID NO:231 is the Cas-alpha 11 sgRNA version 2 RNA sequence        from Artificial.    -   SEQ ID NO:232 is the Cas-alpha 11 sgRNA version 3 RNA sequence        from Artificial.    -   SEQ ID NO:233 is the Cas-alpha 11 sgRNA version 4 RNA sequence        from Artificial.    -   SEQ ID NO:234 is the Cas-alpha 11 sgRNA version 5 RNA sequence        from Artificial.    -   SEQ ID NO:235 is the Cas-alpha 4 Zea mays codon optimized gene        DNA sequence from Artificial.    -   SEQ ID NO:236 is the Cas-alpha 10 Zea mays codon optimized gene        DNA sequence from Artificial.    -   SEQ ID NO:237 is the Cas-alpha 10 Saccharomyces cerevisiae codon        optimized gene DNA sequence from Artificial.    -   SEQ ID NO:238 is the Cas-alpha 4 sgRNA backbone RNA sequence        from Artificial.    -   SEQ ID NO:239 is the Cas-alpha 10 sgRNA backbone RNA sequence        from Artificial.    -   SEQ ID NO:240 is the Cas-alpha 4 Liguleless 2 sgRNA Target        Sequence RNA sequence from Artificial.    -   SEQ ID NO:241 is the Cas-alpha 4 Liguleless 3 sgRNA Target        Sequence RNA sequence from Artificial.    -   SEQ ID NO:242 is the Cas-alpha 10 nptII sgRNA Target Sequence        RNA sequence from Artificial.    -   SEQ ID NO:243 is the Cas-alpha 10 ms26 sgRNA Target Sequence RNA        sequence from Artificial.    -   SEQ ID NO:244 is the Cas-alpha 10 ade2 sgRNA Target Sequence RNA        sequence from Artificial.    -   SEQ ID NO:245 is the Cas-alpha 4 VEGFA 2 sgRNA Target Sequence        RNA sequence from Artificial.    -   SEQ ID NO:246 is the Cas-alpha 4 VEGFA 3 sgRNA Target Sequence        RNA sequence from Artificial.    -   SEQ ID NO:247 is the Cas-alpha 4 sgRNA Targeting Liguleless 2        RNA sequence from Artificial.    -   SEQ ID NO:248 is the Cas-alpha 4 sgRNA Targeting Liguleless 3        RNA sequence from Artificial.    -   SEQ ID NO:249 is the Cas-alpha 10 sgRNA Targeting nptII RNA        sequence from Artificial.    -   SEQ ID NO:250 is the Cas-alpha 10 sgRNA Targeting ms26 RNA        sequence from Artificial.    -   SEQ ID NO:251 is the Cas-alpha 10 sgRNA Targeting ade2 RNA        sequence from Artificial.    -   SEQ ID NO:252 is the Cas-alpha 4 sgRNA Targeting VEGFA 2 RNA        sequence from Artificial.    -   SEQ ID NO:253 is the Cas-alpha 4 sgRNA Targeting VEGFA 3 RNA        sequence from Artificial.    -   SEQ ID NO:254 is the Cas-alpha 12 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:255 is the Cas-alpha 13 endonuclease PRT sequence from        Clostridium paraputrificum.    -   SEQ ID NO:256 is the Cas-alpha 14 endonuclease PRT sequence from        Clostridium novyi.    -   SEQ ID NO:257 is the Cas-alpha 15 endonuclease PRT sequence from        Ruminococcus albus.    -   SEQ ID NO:258 is the Cas-alpha 16 endonuclease PRT sequence from        Clostridium hiranonis.    -   SEQ ID NO:259 is the Cas-alpha 17 endonuclease PRT sequence from        Clostridium ihumii.    -   SEQ ID NO:260 is the Cas-alpha 18 endonuclease PRT sequence from        Cellulosilyticum ruminicola.    -   SEQ ID NO:261 is the Cas-alpha 19 endonuclease PRT sequence from        Eubacterium siraeum.    -   SEQ ID NO:262 is the Cas-alpha 20 endonuclease PRT sequence from        Clostridium botulinum.    -   SEQ ID NO:263 is the Cas-alpha 21 endonuclease PRT sequence from        Clostridium botulinum.    -   SEQ ID NO:264 is the Cas-alpha 22 endonuclease PRT sequence from        Ruminiclostridium hungatei.    -   SEQ ID NO:265 is the Cas-alpha 23 endonuclease PRT sequence from        Desulfovibrio fructosivorans.    -   SEQ ID NO:266 is the Cas-alpha 24 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:267 is the Cas-alpha 25 endonuclease PRT sequence from        Clostridium paraputrificum.    -   SEQ ID NO:268 is the Cas-alpha 26 endonuclease PRT sequence from        Clostridium ventriculi.    -   SEQ ID NO:269 is the Cas-alpha 27 endonuclease PRT sequence from        Ruminococcus sp.    -   SEQ ID NO:270 is the Cas-alpha 28 endonuclease PRT sequence from        Ruminococcus sp.    -   SEQ ID NO:271 is the Cas-alpha 29 endonuclease PRT sequence from        Peptoclostridium sp.    -   SEQ ID NO:272 is the Cas-alpha 30 endonuclease PRT sequence from        Bacillus sp.    -   SEQ ID NO:273 is the Cas-alpha 31 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:274 is the Cas-alpha 32 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:275 is the Cas-alpha 33 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:276 is the Cas-alpha 34 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:277 is the Cas-alpha 35 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:278 is the Cas-alpha 36 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:279 is the Cas-alpha 37 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:280 is the Cas-alpha 38 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:281 is the Cas-alpha 39 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:282 is the Cas-alpha 40 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:283 is the Cas-alpha 41 endonuclease PRT sequence from        uncultured archaeon.    -   SEQ ID NO:284 is the Cas-alpha 42 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:285 is the Cas-alpha 43 endonuclease PRT sequence from        Desulfovibrio fructosivorans.    -   SEQ ID NO:286 is the Cas-alpha 44 endonuclease PRT sequence from        Clostridium botulinum.    -   SEQ ID NO:287 is the Cas-alpha 45 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:288 is the Cas-alpha 46 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:289 is the Cas-alpha 47 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:290 is the Cas-alpha 48 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:291 is the Cas-alpha 49 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:292 is the Cas-alpha 50 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:293 is the Cas-alpha 51 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:294 is the Cas-alpha 52 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:295 is the Cas-alpha 53 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:296 is the Cas-alpha 54 endonuclease PRT sequence from        Clostridioides difficile.    -   SEQ ID NO:297 is the Cas-alpha 55 endonuclease PRT sequence from        Clostridium hiranonis.    -   SEQ ID NO:298 is the Cas-alpha 56 endonuclease PRT sequence from        Clostridioides difficile    -   SEQ ID NO:299 is the Cas-alpha 57 endonuclease PRT sequence from        Aneurinibacillus danicus.    -   SEQ ID NO:300 is the Cas-alpha 58 endonuclease PRT sequence from        Parageobacillus thermoglucosidasius.    -   SEQ ID NO:301 is the Cas-alpha 59 endonuclease PRT sequence from        Brevibacillus centrosporus.    -   SEQ ID NO:302 is the Cas-alpha 60 endonuclease PRT sequence from        Clostridium pasteurianum.    -   SEQ ID NO:303 is the Cas-alpha 61 endonuclease PRT sequence from        Eubacterium siraeum.    -   SEQ ID NO:304 is the Cas-alpha 62 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:305 is the Cas-alpha 63 endonuclease PRT sequence from        Ruminococcus sp.    -   SEQ ID NO:306 is the Cas-alpha 64 endonuclease PRT sequence from        Ruminococcus sp.    -   SEQ ID NO:307 is the Cas-alpha 65 endonuclease PRT sequence from        Clostridium perfringens.    -   SEQ ID NO:308 is the Cas-alpha 66 endonuclease PRT sequence from        Bacillus thuringiensis.    -   SEQ ID NO:309 is the Cas-alpha 67 endonuclease PRT sequence from        Clostridium perfringens.    -   SEQ ID NO:310 is the Cas-alpha 68 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:311 is the Cas-alpha 69 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:312 is the Cas-alpha 70 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:313 is the Cas-alpha 71 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:314 is the Cas-alpha 72 endonuclease PRT sequence from        Alicyclobacillus acidoterrestris.    -   SEQ ID NO:315 is the Cas-alpha 73 endonuclease PRT sequence from        Clostridium tetani.    -   SEQ ID NO:316 is the Cas-alpha 74 endonuclease PRT sequence from        Candidatus Levybacteria bacterium.    -   SEQ ID NO:317 is the Cas-alpha 75 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:318 is the Cas-alpha 76 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:319 is the Cas-alpha 77 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:320 is the Cas-alpha 78 endonuclease PRT sequence from        Clostridium paraputrificum.    -   SEQ ID NO:321 is the Cas-alpha 79 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:322 is the Cas-alpha 80 endonuclease PRT sequence from        Bacillus thuringiensis.    -   SEQ ID NO:323 is the Cas-alpha 81 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:324 is the Cas-alpha 82 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:325 is the Cas-alpha 83 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:326 is the Cas-alpha 84 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:327 is the Cas-alpha 85 endonuclease PRT sequence from        Bacillus wiedmannii.    -   SEQ ID NO:328 is the Cas-alpha 86 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:329 is the Cas-alpha 87 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:330 is the Cas-alpha 88 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:331 is the Cas-alpha 89 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:332 is the Cas-alpha 90 endonuclease PRT sequence from        Bacillus toyonensis.    -   SEQ ID NO:333 is the Cas-alpha 91 endonuclease PRT sequence from        Bacillus thuringiensis.    -   SEQ ID NO:334 is the Cas-alpha 92 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:335 is the Cas-alpha 93 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:336 is the Cas-alpha 94 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:337 is the Cas-alpha 95 endonuclease PRT sequence from        Bacillus thuringiensis.    -   SEQ ID NO:338 is the Cas-alpha 96 endonuclease PRT sequence from        Bacillus sp.    -   SEQ ID NO:339 is the Cas-alpha 97 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:340 is the Cas-alpha 98 endonuclease PRT sequence from        Bacillus cereus.    -   SEQ ID NO:341 is the Cas-alpha 99 endonuclease PRT sequence from        Bacillus thuringiensis.    -   SEQ ID NO:342 is the Cas-alpha 100 endonuclease PRT sequence        from Bacillus sp.    -   SEQ ID NO:343 is the Cas-alpha 101 endonuclease PRT sequence        from Prevotella copri.    -   SEQ ID NO:344 is the Cas-alpha 102 endonuclease PRT sequence        from Prevotella copri.    -   SEQ ID NO:345 is the Cas-alpha 103 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:346 is the Cas-alpha 104 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:347 is the Cas-alpha 105 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:348 is the Cas-alpha 106 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:349 is the Cas-alpha 107 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:350 is the Cas-alpha 108 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:351 is the Cas-alpha 109 endonuclease PRT sequence        from Clostridioides difficile.    -   SEQ ID NO:352 is the Cas-alpha 110 endonuclease PRT sequence        from Flavobacterium thermophilum.    -   SEQ ID NO:353 is the Cas-alpha 111 endonuclease PRT sequence        from Phascolarctobacterium sp.    -   SEQ ID NO:354 is the Cas-alpha 112 endonuclease PRT sequence        from Bacillus pseudomycoides.    -   SEQ ID NO:355 is the Cas-alpha 113 endonuclease PRT sequence        from Bacteroides plebeius.    -   SEQ ID NO:356 is the Cas-alpha 114 endonuclease PRT sequence        from Clostridium botulinum.    -   SEQ ID NO:357 is the Cas-alpha 115 endonuclease PRT sequence        from Bacillus pseudomycoides.    -   SEQ ID NO:358 is the Cas-alpha 116 endonuclease PRT sequence        from Bacillus pseudomycoides.    -   SEQ ID NO:359 is the Cas-alpha 117 endonuclease PRT sequence        from Clostridium botulinum.    -   SEQ ID NO:360 is the Cas-alpha 118 endonuclease PRT sequence        from Clostridium botulinum.    -   SEQ ID NO:361 is the Cas-alpha 119 endonuclease PRT sequence        from Clostridium botulinum.    -   SEQ ID NO:362 is the Cas-alpha 120 endonuclease PRT sequence        from Hydrogenivirga sp.    -   SEQ ID NO:363 is the Cas-alpha 121 endonuclease PRT sequence        from Bacillus megaterium.    -   SEQ ID NO:364 is the Cas-alpha 122 endonuclease PRT sequence        from Clostridium fallax.    -   SEQ ID NO:365 is the Cas-alpha 123 endonuclease PRT sequence        from Bacteroides plebeius.    -   SEQ ID NO:366 is the Cas-alpha 124 endonuclease PRT sequence        from Bacillus thuringiensis.    -   SEQ ID NO:367 is the Cas-alpha 125 endonuclease PRT sequence        from Bacillus cereus.    -   SEQ ID NO:368 is the Cas-alpha 126 endonuclease PRT sequence        from Clostridium sp.    -   SEQ ID NO:369 is the Cas-alpha 127 endonuclease PRT sequence        from Bacteroides plebeius.    -   SEQ ID NO:370 is the Cas-alpha 128 endonuclease PRT sequence        from Dorea longicatena.    -   SEQ ID NO:371 is the Cas-alpha 129 endonuclease PRT sequence        from Sulfurihydrogenibium azorense.

DETAILED DESCRIPTION

Compositions and methods are provided for novel CRISPR effector systemsand elements comprising such systems, including, but not limiting to,novel guide polynucleotide/endonuclease complexes, guidepolynucleotides, guide RNA elements, Cas proteins, and endonucleases, aswell as proteins comprising an endonuclease functionality (domain).Compositions and methods are also provided for direct delivery ofendonucleases, cleavage ready complexes, guide RNAs, and guide RNA/Casendonuclease complexes. The present disclosure further includescompositions and methods for genome modification of a target sequence inthe genome of a cell, for gene editing, and for inserting apolynucleotide of interest into the genome of a cell.

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. It must be noted that, as used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

Definitions

As used herein, “nucleic acid” means a polynucleotide and includes asingle or a double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” and “nucleic acid fragment” are usedinterchangeably to denote a polymer of RNA and/or DNA and/or RNA-DNAthat is single- or double-stranded, optionally comprising synthetic,non-natural, or altered nucleotide bases. Nucleotides (usually found intheir 5′-monophosphate form) are referred to by their single letterdesignation as follows:“A” for adenosine or deoxyadenosine (for RNA orDNA, respectively), “C” for cytosine or deoxycytosine, “G” for guanosineor deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” forpurines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” forA or C or T, “I” for inosine, and “N” for any nucleotide.

The term “genome” as it applies to a prokaryotic and eukaryotic cell ororganism cells encompasses not only chromosomal DNA found within thenucleus, but organelle DNA found within subcellular components (e.g.,mitochondria, or plastid) of the cell.

“Open reading frame” is abbreviated ORF.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence in an in vitro hybridization assay.Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength. Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salt(s)) at pH 7.0 to 8.3, and atleast about 30° C. for short probes (e.g., 10 to 50 nucleotides) and atleast about 60° C. for long probes (e.g., greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringencyconditions include hybridization with a buffer solution of 30 to 35%formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and awash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to55° C. Exemplary moderate stringency conditions include hybridization in40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to1×SSC at 55 to 60° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a washin 0.1×SSC at 60 to 65° C.

By “homology” is meant DNA sequences that are similar. For example, a“region of homology to a genomic region” that is found on the donor DNAis a region of DNA that has a similar sequence to a given “genomicregion” in the cell or organism genome. A region of homology can be ofany length that is sufficient to promote homologous recombination at thecleaved target site. For example, the region of homology can comprise atleast 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60,5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400,5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300,5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200,5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000, 5-3100or more bases in length such that the region of homology has sufficienthomology to undergo homologous recombination with the correspondinggenomic region. “Sufficient homology” indicates that two polynucleotidesequences have sufficient structural similarity to act as substrates fora homologous recombination reaction. The structural similarity includesoverall length of each polynucleotide fragment, as well as the sequencesimilarity of the polynucleotides. Sequence similarity can be describedby the percent sequence identity over the whole length of the sequences,and/or by conserved regions comprising localized similarities such ascontiguous nucleotides having 100% sequence identity, and percentsequence identity over a portion of the length of the sequences.

As used herein, a “genomic region” is a segment of a chromosome in thegenome of a cell that is present on either side of the target site or,alternatively, also comprises a portion of the target site. The genomicregion can comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40,5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100,5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100,5-1200, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000,5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900,5-3000, 5-3100 or more bases such that the genomic region has sufficienthomology to undergo homologous recombination with the correspondingregion of homology.

As used herein, “homologous recombination” (HR) includes the exchange ofDNA fragments between two DNA molecules at the sites of homology. Thefrequency of homologous recombination is influenced by a number offactors. Different organisms vary with respect to the amount ofhomologous recombination and the relative proportion of homologous tonon-homologous recombination. Generally, the length of the region ofhomology affects the frequency of homologous recombination events:thelonger the region of homology, the greater the frequency. The length ofthe homology region needed to observe homologous recombination is alsospecies-variable. In many cases, at least 5 kb of homology has beenutilized, but homologous recombination has been observed with as littleas 25-50 bp of homology. See, for example, Singer et al., (1982) Cell31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al.,(1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992)Mol Cell Biol 12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203;Liskay et al., (1987) Genetics 115:161-7.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

The term “percentage of sequence identity” refers to the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the results by 100to yield the percentage of sequence identity. Useful examples of percentsequence identities include, but are not limited to, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%.These identities can be determined using any of the programs describedherein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, (1989) CABIOS5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191) andfound in the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). For multiple alignments, thedefault values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10.Default parameters for pairwise alignments and calculation of percentidentity of protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=S and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After alignment of the sequences using the Clustal V program, it ispossible to obtain a “percent identity” by viewing the “sequencedistances” Table in the same program. The “Clustal W method ofalignment” corresponds to the alignment method labeled Clustal W(described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins etal., (1992) Comput Appl Biosci 8:189-191) and found in the MegAlign™v6.1 program of the LASERGENE bioinformatics computing suite (DNASTARInc., Madison, Wis.). Default parameters for multiple alignment (GAPPENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNATransition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA WeightMatrix=IUB). After alignment of the sequences using the Clustal Wprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” Table in the same program. Unless otherwise stated,sequence identity/similarity values provided herein refer to the valueobtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) usingthe following parameters: % identity and % similarity for a nucleotidesequence using a gap creation penalty weight of 50 and a gap lengthextension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using a GAPcreation penalty weight of 8 and a gap length extension penalty of 2,and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc.Natl. Acad. Sci. USA 89:10915). GAP uses the algorithm of Needleman andWunsch, (1970) J Mol Biol 48:443-53, to find an alignment of twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps. GAP considers all possible alignments and gappositions and creates the alignment with the largest number of matchedbases and the fewest gaps, using a gap creation penalty and a gapextension penalty in units of matched bases. “BLAST” is a searchingalgorithm provided by the National Center for Biotechnology Information(NCBI) used to find regions of similarity between biological sequences.The program compares nucleotide or protein sequences to sequencedatabases and calculates the statistical significance of matches toidentify sequences having sufficient similarity to a query sequence suchthat the similarity would not be predicted to have occurred randomly.BLAST reports the identified sequences and their local alignment to thequery sequence. It is well understood by one skilled in the art thatmany levels of sequence identity are useful in identifying polypeptidesfrom other species or modified naturally or synthetically wherein suchpolypeptides have the same or similar function or activity. Usefulexamples of percent identities include, but are not limited to, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from50% to 100%. Indeed, any amino acid identity from 50% to 100% may beuseful in describing the present disclosure, such as 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%.

Polynucleotide and polypeptide sequences, variants thereof, and thestructural relationships of these sequences can be described by theterms “homology”, “homologous”, “substantially identical”,“substantially similar” and “corresponding substantially” which are usedinterchangeably herein. These refer to polypeptide or nucleic acidsequences wherein changes in one or more amino acids or nucleotide basesdo not affect the function of the molecule, such as the ability tomediate gene expression or to produce a certain phenotype. These termsalso refer to modification(s) of nucleic acid sequences that do notsubstantially alter the functional properties of the resulting nucleicacid relative to the initial, unmodified nucleic acid. Thesemodifications include deletion, substitution, and/or insertion of one ormore nucleotides in the nucleic acid fragment. Substantially similarnucleic acid sequences encompassed may be defined by their ability tohybridize (under moderately stringent conditions, e.g., 0.5×SSC, 0.1%SDS, 60° C.) with the sequences exemplified herein, or to any portion ofthe nucleotide sequences disclosed herein and which are functionallyequivalent to any of the nucleic acid sequences disclosed herein.Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

A “centimorgan” (cM) or “map unit” is the distance between twopolynucleotide sequences, linked genes, markers, target sites, loci, orany pair thereof, wherein 1% of the products of meiosis are recombinant.Thus, a centimorgan is equivalent to a distance equal to a 1% averagerecombination frequency between the two linked genes, markers, targetsites, loci, or any pair thereof.

An “isolated” or “purified” nucleic acid molecule, polynucleotide,polypeptide, or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or polypeptide or protein is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived. Isolated polynucleotidesmay be purified from a cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The term “fragment” refers to a contiguous set of nucleotides or aminoacids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20 contiguousnucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20 contiguousamino acids. A fragment may or may not exhibit the function of asequence sharing some percent identity over the length of said fragment.

The terms “fragment that is functionally equivalent” and “functionallyequivalent fragment” are used interchangeably herein. These terms referto a portion or subsequence of an isolated nucleic acid fragment orpolypeptide that displays the same activity or function as the longersequence from which it derives. In one example, the fragment retains theability to alter gene expression or produce a certain phenotype whetheror not the fragment encodes an active protein. For example, the fragmentcan be used in the design of genes to produce the desired phenotype in amodified plant. Genes can be designed for use in suppression by linkinga nucleic acid fragment, whether or not it encodes an active enzyme, inthe sense or antisense orientation relative to a plant promotersequence.

“Gene” includes a nucleic acid fragment that expresses a functionalmolecule such as, but not limited to, a specific protein, includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in its natural endogenous location with its own regulatorysequences.

By the term “endogenous” it is meant a sequence or other molecule thatnaturally occurs in a cell or organism. In one aspect, an endogenouspolynucleotide is normally found in the genome of a cell; that is, notheterologous.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same, that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differ,that plant is heterozygous at that locus.

“Coding sequence” refers to a polynucleotide sequence which codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences include, but arenot limited to, promoters, translation leader sequences, 5′ untranslatedsequences, 3′ untranslated sequences, introns, polyadenylation targetsequences, RNA processing sites, effector binding sites, and stem-loopstructures.

A “mutated gene” is a gene that has been altered through humanintervention. Such a “mutated gene” has a sequence that differs from thesequence of the corresponding non-mutated gene by at least onenucleotide addition, deletion, or substitution. In certain embodimentsof the disclosure, the mutated gene comprises an alteration that resultsfrom a guide polynucleotide/Cas endonuclease system as disclosed herein.A mutated plant is a plant comprising a mutated gene.

As used herein, a “targeted mutation” is a mutation in a gene (referredto as the target gene), including a native gene, that was made byaltering a target sequence within the target gene using any method knownto one skilled in the art, including a method involving a guided Casendonuclease system as disclosed herein.

The terms “knock-out”, “gene knock-out” and “genetic knock-out” are usedinterchangeably herein. A knock-out represents a DNA sequence of a cellthat has been rendered partially or completely inoperative by targetingwith a Cas protein; for example, a DNA sequence prior to knock-out couldhave encoded an amino acid sequence, or could have had a regulatoryfunction (e.g., promoter).

The terms “knock-in”, “gene knock-in, “gene insertion” and “geneticknock-in” are used interchangeably herein. A knock-in represents thereplacement or insertion of a DNA sequence at a specific DNA sequence incell by targeting with a Cas protein (for example by homologousrecombination (HR), wherein a suitable donor DNA polynucleotide is alsoused). examples of knock-ins are a specific insertion of a heterologousamino acid coding sequence in a coding region of a gene, or a specificinsertion of a transcriptional regulatory element in a genetic locus.

By “domain” it is meant a contiguous stretch of nucleotides (that can beRNA, DNA, and/or RNA-DNA-combination sequence) or amino acids.

The term “conserved domain” or “motif” means a set of polynucleotides oramino acids conserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialto the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

A “codon-modified gene” or “codon-preferred gene” or “codon-optimizedgene” is a gene having its frequency of codon usage designed to mimicthe frequency of preferred codon usage of the host cell.

An “optimized” polynucleotide is a sequence that has been optimized forimproved expression in a particular heterologous host cell.

A “plant-optimized nucleotide sequence” is a nucleotide sequence thathas been optimized for expression in plants, particularly for increasedexpression in plants. A plant-optimized nucleotide sequence includes acodon-optimized gene. A plant-optimized nucleotide sequence can besynthesized by modifying a nucleotide sequence encoding a protein suchas, for example, a Cas endonuclease as disclosed herein, using one ormore plant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage.

A “promoter” is a region of DNA involved in recognition and binding ofRNA polymerase and other proteins to initiate transcription. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers. An“enhancer” is a DNA sequence that can stimulate promoter activity, andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue-specificity of a promoter.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, and/or comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of some variation may have identical promoter activity.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. The term“inducible promoter” refers to a promoter that selectively express acoding sequence or functional RNA in response to the presence of anendogenous or exogenous stimulus, for example by chemical compounds(chemical inducers) or in response to environmental, hormonal, chemical,and/or developmental signals. Inducible or regulated promoters include,for example, promoters induced or regulated by light, heat, stress,flooding or drought, salt stress, osmotic stress, phytohormones,wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonate,salicylic acid, or safeners.

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences havebeen described (e.g., Turner and Foster, (1995) Mol Biotechnol3:225-236).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht et al., (1989) Plant Cell1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complimentary copy of the DNA sequence, it isreferred to as the primary transcript or pre-mRNA. A RNA transcript isreferred to as the mature RNA or mRNA when it is a RNA sequence derivedfrom post-transcriptional processing of the primary transcript pre-mRNA.“Messenger RNA” or “mRNA” refers to the RNA that is without introns andthat can be translated into protein by the cell. “cDNA” refers to a DNAthat is complementary to, and synthesized from, an mRNA template usingthe enzyme reverse transcriptase. The cDNA can be single-stranded orconverted into double-stranded form using the Klenow fragment of DNApolymerase I. “Sense” RNA refers to RNA transcript that includes themRNA and can be translated into protein within a cell or in vitro.“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA, and that blocks theexpression of a target gene (see, e.g., U.S. Pat. No. 5,107,065). Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e., at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that may not be translated butyet has an effect on cellular processes. The terms “complement” and“reverse complement” are used interchangeably herein with respect tomRNA transcripts, and are meant to define the antisense RNA of themessage.

The term “genome” refers to the entire complement of genetic material(genes and non-coding sequences) that is present in each cell of anorganism, or virus or organelle; and/or a complete set of chromosomesinherited as a (haploid) unit from one parent.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions can beoperably linked, either directly or indirectly, 5′ to the target mRNA,or 3′ to the target mRNA, or within the target mRNA, or a firstcomplementary region is 5′ and its complement is 3′ to the target mRNA.

Generally, “host” refers to an organism or cell into which aheterologous component (polynucleotide, polypeptide, other molecule,cell) has been introduced. As used herein, a “host cell” refers to an invivo or in vitro eukaryotic cell, prokaryotic cell (e.g., bacterial orarchaeal cell), or cell from a multicellular organism (e.g., a cellline) cultured as a unicellular entity, into which a heterologouspolynucleotide or polypeptide has been introduced. In some embodiments,the cell is selected from the group consisting of: an archaeal cell, abacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, asomatic cell, a germ cell, a stem cell, a plant cell, an algal cell, ananimal cell, in invertebrate cell, a vertebrate cell, a fish cell, afrog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, acow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mousecell, a non-human primate cell, and a human cell. In some cases, thecell is in vitro. In some cases, the cell is in vivo.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis,or manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to a linear orcircular extra chromosomal element often carrying genes that are notpart of the central metabolism of the cell, and usually in the form ofdouble-stranded DNA. Such elements may be autonomously replicatingsequences, genome integrating sequences, phage, or nucleotide sequences,in linear or circular form, of a single- or double-stranded DNA or RNA,derived from any source, in which a number of nucleotide sequences havebeen joined or recombined into a unique construction which is capable ofintroducing a polynucleotide of interest into a cell. “Transformationcassette” refers to a specific vector comprising a gene and havingelements in addition to the gene that facilitates transformation of aparticular host cell. “Expression cassette” refers to a specific vectorcomprising a gene and having elements in addition to the gene that allowfor expression of that gene in a host.

The terms “recombinant DNA molecule”, “recombinant DNA construct”,“expression construct”, “construct”, and “recombinant construct” areused interchangeably herein. A recombinant DNA construct comprises anartificial combination of nucleic acid sequences, e.g., regulatory andcoding sequences that are not all found together in nature. For example,a recombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that will be used to introduce the vector into the host cellsas is well known to those skilled in the art. For example, a plasmidvector can be used. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells. The skilled artisan willalso recognize that different independent transformation events mayresult in different levels and patterns of expression (Jones et al.,(1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics218:78-86), and thus that multiple events are typically screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished standard molecularbiological, biochemical, and other assays including Southern analysis ofDNA, Northern analysis of mRNA expression, PCR, real time quantitativePCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting analysisof protein expression, enzyme or activity assays, and/or phenotypicanalysis.

The term “heterologous” refers to the difference between the originalenvironment, location, or composition of a particular polynucleotide orpolypeptide sequence and its current environment, location, orcomposition. Non-limiting examples include differences in taxonomicderivation (e.g., a polynucleotide sequence obtained from Zea mays wouldbe heterologous if inserted into the genome of an Oryza sativa plant, orof a different variety or cultivar of Zea mays; or a polynucleotideobtained from a bacterium was introduced into a cell of a plant), orsequence (e.g., a polynucleotide sequence obtained from Zea mays,isolated, modified, and re-introduced into a maize plant). As usedherein, “heterologous” in reference to a sequence can refer to asequence that originates from a different species, variety, foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention. For example, a promoter operably linked to a heterologouspolynucleotide is from a species different from the species from whichthe polynucleotide was derived, or, if from the same/analogous species,one or both are substantially modified from their original form and/orgenomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide. Alternatively, one or more regulatoryregion(s) and/or a polynucleotide provided herein may be entirelysynthetic. In another example, a target polynucleotide for cleavage by aCas endonuclease may be of a different organism than that of the Casendonuclease. In another example, a Cas endonuclease and guide RNA maybe introduced to a target polynucleotide with an additionalpolynucleotide that acts as a template or donor for insertion into thetarget polynucleotide, wherein the additional polynucleotide isheterologous to the target polynucleotide and/or the Cas endonuclease.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., an mRNA, guide RNA, or a protein) ineither precursor or mature form.

A “mature” protein refers to a post-translationally processedpolypeptide (i.e., one from which any pre- or propeptides present in theprimary translation product have been removed).

“Precursor” protein refers to the primary product of translation of mRNA(i.e., with pre- and propeptides still present). Pre- and propeptidesmay be but are not limited to intracellular localization signals.

“CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats)loci refers to certain genetic loci encoding components of DNA cleavagesystems, for example, used by bacterial and archaeal cells to destroyforeign DNA (Horvath and Barrangou, 2010, Science 327:167-170;WO2007025097, published 1 Mar. 2007). A CRISPR locus can consist of aCRISPR array, comprising short direct repeats (CRISPR repeats) separatedby short variable DNA sequences (called spacers), which can be flankedby diverse Cas (CRISPR-associated) genes.

As used herein, an “effector” or “effector protein” is a protein thatencompasses an activity including recognizing, binding to, and/orcleaving or nicking a polynucleotide target. An effector, or effectorprotein, may also be an endonuclease. The “effector complex” of a CRISPRsystem includes Cas proteins involved in crRNA and target recognitionand binding. Some of the component Cas proteins may additionallycomprise domains involved in target polynucleotide cleavage.

The term “Cas protein” refers to a polypeptide encoded by a Cas(CRISPR-associated) gene. A Cas protein includes proteins encoded by agene in a cas locus, and include adaptation molecules as well asinterference molecules. An interference molecule of a bacterial adaptiveimmunity complex includes endonucleases. A Cas endonuclease describedherein comprises one or more nuclease domains. A Cas endonucleaseincludes but is not limited to: the novel Cas-alpha protein disclosedherein, a Cas9 protein, a Cpf1 (Cas12) protein, a C2c1 protein, a C2c2protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10, orcombinations or complexes of these. A Cas protein may be a “Casendonuclease” or “Cas effector protein”, that when in complex with asuitable polynucleotide component, is capable of recognizing, bindingto, and optionally nicking or cleaving all or part of a specificpolynucleotide target sequence. The Cas-alpha endonucleases of thedisclosure include those having one or more RuvC nuclease domains. A Casprotein is further defined as a functional fragment or functionalvariant of a native Cas protein, or a protein that shares at least 50%,between 50% and 55%, at least 55%, between 55% and 60%, at least 60%,between 60% and 65%, at least 65%, between 65% and 70%, at least 70%,between 70% and 75%, at least 75%, between 75% and 80%, at least 80%,between 80% and 85%, at least 85%, between 85% and 90%, at least 90%,between 90% and 95%, at least 95%, between 95% and 96%, at least 96%,between 96% and 97%, at least 97%, between 97% and 98%, at least 98%,between 98% and 99%, at least 99%, between 99% and 100%, or 100%sequence identity with at least 50, between 50 and 100, at least 100,between 100 and 150, at least 150, between 150 and 200, at least 200,between 200 and 250, at least 250, between 250 and 300, at least 300,between 300 and 350, at least 350, between 350 and 400, at least 400,between 400 and 450, at least 500, or greater than 500 contiguous aminoacids of a native Cas protein, and retains at least partial activity ofthe native sequence.

A “functional fragment”, “fragment that is functionally equivalent” and“functionally equivalent fragment” of a Cas endonuclease are usedinterchangeably herein, and refer to a portion or subsequence of the Casendonuclease of the present disclosure in which the ability torecognize, bind to, and optionally unwind, nick or cleave (introduce asingle or double-strand break in) the target site is retained. Theportion or subsequence of the Cas endonuclease can comprise a completeor partial (functional) peptide of any one of its domains such as forexample, but not limiting to a complete of functional part of a Cas3 HDdomain, a complete of functional part of a Cas3 Helicase domain,complete of functional part of a protein (such as but not limiting to aCas5, Cas5d, Cas? and Cas8b1).

The terms “functional variant”, “variant that is functionallyequivalent” and “functionally equivalent variant” of a Cas endonucleaseor Cas effector protein, including Cas-alpha described herein, are usedinterchangeably herein, and refer to a variant of the Cas effectorprotein disclosed herein in which the ability to recognize, bind to, andoptionally unwind, nick or cleave all or part of a target sequence isretained.

A Cas endonuclease may also include a multifunctional Cas endonuclease.The term “multifunctional Cas endonuclease” and “multifunctional Casendonuclease polypeptide” are used interchangeably herein and includesreference to a single polypeptide that has Cas endonucleasefunctionality (comprising at least one protein domain that can act as aCas endonuclease) and at least one other functionality, such as but notlimited to, the functionality to form a complex (comprises at least asecond protein domain that can form a complex with other proteins). Inone aspect, the multifunctional Cas endonuclease comprises at least oneadditional protein domain relative (either internally, upstream (5′),downstream (3′), or both internally 5′ and 3′, or any combinationthereof) to those domains typical of a Cas endonuclease.

The terms “cascade” and “cascade complex” are used interchangeablyherein and include reference to a multi-subunit protein complex that canassemble with a polynucleotide forming a polynucleotide-protein complex(PNP). Cascade is a PNP that relies on the polynucleotide for complexassembly and stability, and for the identification of target nucleicacid sequences. Cascade functions as a surveillance complex that findsand optionally binds target nucleic acids that are complementary to avariable targeting domain of the guide polynucleotide.

The terms “cleavage-ready Cascade”, “crCascade”, “cleavage-ready Cascadecomplex”, “crCascade complex”, “cleavage-ready Cascade system”, “CRC”and “crCascade system”, are used interchangeably herein and includereference to a multi-subunit protein complex that can assemble with apolynucleotide forming a polynucleotide-protein complex (PNP), whereinone of the cascade proteins is a Cas endonuclease capable ofrecognizing, binding to, and optionally unwinding, nicking, or cleavingall or part of a target sequence.

The terms “5′-cap” and “7-methylguanylate (m7G) cap” are usedinterchangeably herein. A 7-methylguanylate residue is located on the 5′terminus of messenger RNA (mRNA) in eukaryotes. RNA polymerase II (PolII) transcribes mRNA in eukaryotes. Messenger RNA capping occursgenerally as follows: the most terminal 5′ phosphate group of the mRNAtranscript is removed by RNA terminal phosphatase, leaving two terminalphosphates. A guanosine monophosphate (GMP) is added to the terminalphosphate of the transcript by a guanylyl transferase, leaving a 5′-5′triphosphate-linked guanine at the transcript terminus. Finally, the7-nitrogen of this terminal guanine is methylated by a methyltransferase.

The terminology “not having a 5′-cap” herein is used to refer to RNAhaving, for example, a 5′-hydroxyl group instead of a 5′-cap. Such RNAcan be referred to as “uncapped RNA”, for example. Uncapped RNA canbetter accumulate in the nucleus following transcription, since5′-capped RNA is subject to nuclear export. One or more RNA componentsherein are uncapped.

As used herein, the term “guide polynucleotide”, relates to apolynucleotide sequence that can form a complex with a Cas endonuclease,including the Cas endonuclease described herein, and enables the Casendonuclease to recognize, optionally bind to, and optionally cleave aDNA target site. The guide polynucleotide sequence can be a RNAsequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence).

The terms “functional fragment”, “fragment that is functionallyequivalent” and “functionally equivalent fragment” of a guide RNA, crRNAor tracrRNA are used interchangeably herein, and refer to a portion orsubsequence of the guide RNA, crRNA or tracrRNA, respectively, of thepresent disclosure in which the ability to function as a guide RNA,crRNA or tracrRNA, respectively, is retained.

The terms “functional variant”, “variant that is functionallyequivalent” and “functionally equivalent variant” of a guide RNA, crRNAor tracrRNA (respectively) are used interchangeably herein, and refer toa variant of the guide RNA, crRNA or tracrRNA, respectively, of thepresent disclosure in which the ability to function as a guide RNA,crRNA or tracrRNA, respectively, is retained.

The terms “single guide RNA” and “sgRNA” are used interchangeably hereinand relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPRRNA) comprising a variable targeting domain (linked to a tracr matesequence that hybridizes to a tracrRNA), fused to a tracrRNA(trans-activating CRISPR RNA). The single guide RNA can comprise a crRNAor crRNA fragment and a tracrRNA or tracrRNA fragment of the type IICRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein said guide RNA/Cas endonuclease complex can directthe Cas endonuclease to a DNA target site, enabling the Cas endonucleaseto recognize, optionally bind to, and optionally nick or cleave(introduce a single or double-strand break) the DNA target site.

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and includes a nucleotide sequence that canhybridize (is complementary) to one strand (nucleotide sequence) of adouble strand DNA target site. The percent complementation between thefirst nucleotide sequence domain (VT domain) and the target sequence canbe at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variabletargeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In someembodiments, the variable targeting domain comprises a contiguousstretch of 12 to 30 nucleotides. The variable targeting domain can becomposed of a DNA sequence, a RNA sequence, a modified DNA sequence, amodified RNA sequence, or any combination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” (of aguide polynucleotide) is used interchangeably herein and includes anucleotide sequence that interacts with a Cas endonuclease polypeptide.A CER domain comprises a (trans-acting) tracrNucleotide mate sequencefollowed by a tracrNucleotide sequence. The CER domain can be composedof a DNA sequence, a RNA sequence, a modified DNA sequence, a modifiedRNA sequence (see for example US20150059010A1, published 26 Feb. 2015),or any combination thereof.

As used herein, the terms “guide polynucleotide/Cas endonucleasecomplex”, “guide polynucleotide/Cas endonuclease system”, “guidepolynucleotide/Cas complex”, “guide polynucleotide/Cas system” and“guided Cas system” “Polynucleotide-guided endonuclease”, “PGEN” areused interchangeably herein and refer to at least one guidepolynucleotide and at least one Cas endonuclease, that are capable offorming a complex, wherein said guide polynucleotide/Cas endonucleasecomplex can direct the Cas endonuclease to a DNA target site, enablingthe Cas endonuclease to recognize, bind to, and optionally nick orcleave (introduce a single or double-strand break) the DNA target site.A guide polynucleotide/Cas endonuclease complex herein can comprise Casprotein(s) and suitable polynucleotide component(s) of any of the knownCRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170;Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetscheet al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60,1-13).

The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Casendonuclease system”, “guide RNA/Cas complex”, “guide RNA/Cas system”,“gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease”, “RGEN”are used interchangeably herein and refer to at least one RNA componentand at least one Cas endonuclease that are capable of forming a complex,wherein said guide RNA/Cas endonuclease complex can direct the Casendonuclease to a DNA target site, enabling the Cas endonuclease torecognize, bind to, and optionally nick or cleave (introduce a single ordouble-strand break) the DNA target site.

The terms “target site”, “target sequence”, “target site sequence,“target DNA”, “target locus”, “genomic target site”, “genomic targetsequence”, “genomic target locus” and “protospacer”, are usedinterchangeably herein and refer to a polynucleotide sequence such as,but not limited to, a nucleotide sequence on a chromosome, episome, alocus, or any other DNA molecule in the genome (including chromosomal,chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which aguide polynucleotide/Cas endonuclease complex can recognize, bind to,and optionally nick or cleave. The target site can be an endogenous sitein the genome of a cell, or alternatively, the target site can beheterologous to the cell and thereby not be naturally occurring in thegenome of the cell, or the target site can be found in a heterologousgenomic location compared to where it occurs in nature. As used herein,terms “endogenous target sequence” and “native target sequence” are usedinterchangeable herein to refer to a target sequence that is endogenousor native to the genome of a cell and is at the endogenous or nativeposition of that target sequence in the genome of the cell. An“artificial target site” or “artificial target sequence” are usedinterchangeably herein and refer to a target sequence that has beenintroduced into the genome of a cell. Such an artificial target sequencecan be identical in sequence to an endogenous or native target sequencein the genome of a cell but be located in a different position (i.e., anon-endogenous or non-native position) in the genome of a cell.

A “protospacer adjacent motif” (PAM) herein refers to a short nucleotidesequence adjacent to a target sequence (protospacer) that is recognized(targeted) by a guide polynucleotide/Cas endonuclease system describedherein. The Cas endonuclease may not successfully recognize a target DNAsequence if the target DNA sequence is not followed by a PAM sequence.The sequence and length of a PAM herein can differ depending on the Casprotein or Cas protein complex used. The PAM sequence can be of anylength but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 nucleotides long.

An “altered target site”, “altered target sequence”, “modified targetsite”, “modified target sequence” are used interchangeably herein andrefer to a target sequence as disclosed herein that comprises at leastone alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, (iv) a chemical alteration of atleast one nucleotide, or (v) any combination of (i)-(iv).

A “modified nucleotide” or “edited nucleotide” refers to a nucleotidesequence of interest that comprises at least one alteration whencompared to its non-modified nucleotide sequence. Such “alterations”include, for example: (i) replacement of at least one nucleotide, (ii) adeletion of at least one nucleotide, (iii) an insertion of at least onenucleotide, (iv) a chemical alteration of at least one nucleotide, or(v) any combination of (i)-(iv).

Methods for “modifying a target site” and “altering a target site” areused interchangeably herein and refer to methods for producing analtered target site.

As used herein, “donor DNA” is a DNA construct that comprises apolynucleotide of interest to be inserted into the target site of a Casendonuclease.

The term “polynucleotide modification template” includes apolynucleotide that comprises at least one nucleotide modification whencompared to the nucleotide sequence to be edited. A nucleotidemodification can be at least one nucleotide substitution, addition ordeletion. Optionally, the polynucleotide modification template canfurther comprise homologous nucleotide sequences flanking the at leastone nucleotide modification, wherein the flanking homologous nucleotidesequences provide sufficient homology to the desired nucleotide sequenceto be edited.

The term “plant-optimized Cas endonuclease” herein refers to a Casprotein, including a multifunctional Cas protein, encoded by anucleotide sequence that has been optimized for expression in a plantcell or plant.

A “plant-optimized nucleotide sequence encoding a Cas endonuclease”,“plant-optimized construct encoding a Cas endonuclease” and a“plant-optimized polynucleotide encoding a Cas endonuclease” are usedinterchangeably herein and refer to a nucleotide sequence encoding a Casprotein, or a variant or functional fragment thereof, that has beenoptimized for expression in a plant cell or plant. A plant comprising aplant-optimized Cas endonuclease includes a plant comprising thenucleotide sequence encoding for the Cas sequence and/or a plantcomprising the Cas endonuclease protein. In one aspect, theplant-optimized Cas endonuclease nucleotide sequence is amaize-optimized, rice-optimized, wheat-optimized, soybean-optimized,cotton-optimized, or canola-optimized Cas endonuclease.

The term “plant” generically includes whole plants, plant organs, planttissues, seeds, plant cells, seeds and progeny of the same. The plant isa monocot or dicot. Plant cells include, without limitation, cells fromseeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen andmicrospores. A “plant element” is intended to reference either a wholeplant or a plant component, which may comprise differentiated and/orundifferentiated tissues, for example but not limited to plant tissues,parts, and cell types. In one embodiment, a plant element is one of thefollowing: whole plant, seedling, meristematic tissue, ground tissue,vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower,fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, andvarious forms of cells and culture (e.g., single cells, protoplasts,embryos, callus tissue). It should be noted that a protoplast is nottechnically an “intact” plant cell (as naturally found with allcomponents), as protoplasts lack a cell wall. The term “plant organ”refers to plant tissue or a group of tissues that constitute amorphologically and functionally distinct part of a plant. As usedherein, a “plant element” is synonymous to a “portion” of a plant, andrefers to any part of the plant, and can include distinct tissues and/ororgans, and may be used interchangeably with the term “tissue”throughout. Similarly, a “plant reproductive element” is intended togenerically reference any part of a plant that is able to initiate otherplants via either sexual or asexual reproduction of that plant, forexample but not limited to: seed, seedling, root, shoot, cutting, scion,graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element maybe in plant or in a plant organ, tissue culture, or cell culture.

“Progeny” comprises any subsequent generation of a plant.

As used herein, the term “plant part” refers to plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers, and the like, as well as the partsthemselves. Grain is intended to mean the mature seed produced bycommercial growers for purposes other than growing or reproducing thespecies. Progeny, variants, and mutants of the regenerated plants arealso included within the scope of the invention, provided that theseparts comprise the introduced polynucleotides.

The term “monocotyledonous” or “monocot” refers to the subclass ofangiosperm plants also known as “monocotyledoneae”, whose seedstypically comprise only one embryonic leaf, or cotyledon. The termincludes references to whole plants, plant elements, plant organs (e.g.,leaves, stems, roots, etc.), seeds, plant cells, and progeny of thesame.

The term “dicotyledonous” or “dicot” refers to the subclass ofangiosperm plants also knows as “dicotyledoneae”, whose seeds typicallycomprise two embryonic leaves, or cotyledons. The term includesreferences to whole plants, plant elements, plant organs (e.g., leaves,stems, roots, etc.), seeds, plant cells, and progeny of the same.

As used herein, a “male sterile plant” is a plant that does not producemale gametes that are viable or otherwise capable of fertilization. Asused herein, a “female sterile plant” is a plant that does not producefemale gametes that are viable or otherwise capable of fertilization. Itis recognized that male-sterile and female-sterile plants can befemale-fertile and male-fertile, respectively. It is further recognizedthat a male fertile (but female sterile) plant can produce viableprogeny when crossed with a female fertile plant and that a femalefertile (but male sterile) plant can produce viable progeny when crossedwith a male fertile plant.

The term “non-conventional yeast” herein refers to any yeast that is nota Saccharomyces (e.g., S. cerevisiae) or Schizosaccharomyces yeastspecies. (see “Non-Conventional Yeasts in Genetics, Biochemistry andBiotechnology: Practical Protocols”, K. Wolf, K. D. Breunig, G. Barth,Eds., Springer-Verlag, Berlin, Germany, 2003).

The term “crossed” or “cross” or “crossing” in the context of thisdisclosure means the fusion of gametes via pollination to produceprogeny (i.e., cells, seeds, or plants). The term encompasses bothsexual crosses (the pollination of one plant by another) and selfing(self-pollination, i.e., when the pollen and ovule (or microspores andmegaspores) are from the same plant or genetically identical plants).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny plant via a sexual cross between twoparent plants, where at least one of the parent plants has the desiredallele within its genome. Alternatively, for example, transmission of anallele can occur by recombination between two donor genomes, e.g., in afused protoplast, where at least one of the donor protoplasts has thedesired allele in its genome. The desired allele can be, e.g., atransgene, a modified (mutated or edited) native allele, or a selectedallele of a marker or QTL.

The term “isoline” is a comparative term, and references organisms thatare genetically identical, but differ in treatment. In one example, twogenetically identical maize plant embryos may be separated into twodifferent groups, one receiving a treatment (such as the introduction ofa CRISPR-Cas effector endonuclease) and one control that does notreceive such treatment. Any phenotypic differences between the twogroups may thus be attributed solely to the treatment and not to anyinherency of the plant's endogenous genetic makeup.

“Introducing” is intended to mean presenting to a target, such as a cellor organism, a polynucleotide or polypeptide or polynucleotide-proteincomplex, in such a manner that the component(s) gains access to theinterior of a cell of the organism or to the cell itself.

A “polynucleotide of interest” includes any nucleotide sequence encodinga protein or polypeptide that improves desirability of crops, i.e. atrait of agronomic interest. Polynucleotides of interest include, butare not limited to: polynucleotides encoding important traits foragronomics, herbicide-resistance, insecticidal resistance, diseaseresistance, nematode resistance, herbicide resistance, microbialresistance, fungal resistance, viral resistance, fertility or sterility,grain characteristics, commercial products, phenotypic marker, or anyother trait of agronomic or commercial importance. A polynucleotide ofinterest may additionally be utilized in either the sense or anti-senseorientation. Further, more than one polynucleotide of interest may beutilized together, or “stacked”, to provide additional benefit.

A “complex trait locus” includes a genomic locus that has multipletransgenes genetically linked to each other.

The compositions and methods herein may provide for an improved“agronomic trait” or “trait of agronomic importance” or “trait ofagronomic interest” to a plant, which may include, but not be limitedto, the following: disease resistance, drought tolerance, heattolerance, cold tolerance, salinity tolerance, metal tolerance,herbicide tolerance, improved water use efficiency, improved nitrogenutilization, improved nitrogen fixation, pest resistance, herbivoreresistance, pathogen resistance, yield improvement, health enhancement,vigor improvement, growth improvement, photosynthetic capabilityimprovement, nutrition enhancement, altered protein content, altered oilcontent, increased biomass, increased shoot length, increased rootlength, improved root architecture, modulation of a metabolite,modulation of the proteome, increased seed weight, altered seedcarbohydrate composition, altered seed oil composition, altered seedprotein composition, altered seed nutrient composition, as compared toan isoline plant not comprising a modification derived from the methodsor compositions herein.

“Agronomic trait potential” is intended to mean a capability of a plantelement for exhibiting a phenotype, preferably an improved agronomictrait, at some point during its life cycle, or conveying said phenotypeto another plant element with which it is associated in the same plant.

The terms “decreased,” “fewer,” “slower” and “increased” “faster”“enhanced” “greater” as used herein refers to a decrease or increase ina characteristic of the modified plant element or resulting plantcompared to an unmodified plant element or resulting plant. For example,a decrease in a characteristic may be at least 1%, at least 2%, at least3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least25%, at least 30%, between 30% and 40%, at least 35%, at least 40%,between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, atleast about 60%, between 60% and 70%, between 70% and 80%, at least 75%,at least about 80%, between 80% and 90%, at least about 90%, between 90%and 100%, at least 100%, between 100% and 200%, at least 200%, at leastabout 300%, at least about 400% or more lower than the untreated controland an increase may be at least 1%, at least 2%, at least 3%, at least4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%,at least 15%, at least 20%, between 20% and 30%, at least 25%, at least30%, between 30% and 40%, at least 35%, at least 40%, between 40% and50%, at least 45%, at least 50%, between 50% and 60%, at least about60%, between 60% and 70%, between 70% and 80%, at least 75%, at leastabout 80%, between 80% and 90%, at least about 90%, between 90% and100%, at least 100%, between 100% and 200%, at least 200%, at leastabout 300%, at least about 400% or more higher than the untreatedcontrol.

As used herein, the term “before”, in reference to a sequence position,refers to an occurrence of one sequence upstream, or 5′, to anothersequence.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” or “umole” mean micromole(s), “g” means gram(s),“μg” or “ug” means microgram(s), “ng” means nanogram(s), “U” meansunit(s), “bp” means base pair(s) and “kb” means kilobase(s).

Classification of CRISPR-Cas Systems

CRISPR-Cas systems have been classified according to sequence andstructural analysis of components. Multiple CRISPR/Cas systems have beendescribed including Class 1 systems, with multisubunit effectorcomplexes (comprising type I, type III, and type IV), and Class 2systems, with single protein effectors (comprising type II, type V, andtype VI) (Makarova et al. 2015, Nature Reviews Microbiology Vol.13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015,Molecular Cell 60, 1-13; Haft et al., 2005, Computational Biology, PLoSComput Biol 1(6):e60; and Koonin et al. 2017, Curr Opinion Microbiology37:67-78).

A CRISPR-Cas system comprises, at a minimum, a CRISPR RNA (crRNA)molecule and at least one CRISPR-associated (Cas) protein to form crRNAribonucleoprotein (crRNP) effector complexes. CRISPR-Cas loci comprisean array of identical repeats interspersed with DNA-targeting spacersthat encode the crRNA components and an operon-like unit of cas genesencoding the Cas protein components. The resulting ribonucleoproteincomplex recognizes a polynucleotide in a sequence-specific manner (Joreet al., Nature Structural & Molecular Biology 18,529-536 (2011)). ThecrRNA serves as a guide RNA for sequence specific binding of theeffector (protein or complex) to double strand DNA sequences, by formingbase pairs with the complementary DNA strand while displacing thenoncomplementary strand to form a so called R-loop. (Jore et al., 2011.Nature Structural & Molecular Biology 18, 529-536).

RNA transcripts of CRISPR loci (pre-crRNA) are cleaved specifically inthe repeat sequences by CRISPR associated (Cas) endoribonucleases intype I and type III systems or by RNase III in type II systems. Thenumber of CRISPR-associated genes at a given CRISPR locus can varybetween species.

Different cas genes that encode proteins with different domains arepresent in different CRISPR systems. The cas operon comprises genes thatencode for one or more effector endonucleases, as well as other Casproteins. Protein subunits include those described in Makarova et al.2011, Nat Rev Microbiol. 2011 9(6):467-477; Makarova et al. 2015, NatureReviews Microbiology Vol. 13:1-15; and Koonin et al. 2017, CurrentOpinion Microbiology 37:67-78). The types of domains include thoseinvolved in Expression (pre-crRNA processing, for example Cas 6 orRNaseIII), Interference (including an effector module for crRNA andtarget binding, as well as domain(s) for target cleavage), Adaptation(spacer insertion, for example Cas1 or Cas2), and Ancillary (regulationor helper or unknown function). Some domains may serve more than onepurpose, for example Cas9 comprises domains for endonucleasefunctionality as well as for target cleavage, among others.

The Cas endonuclease is guided by a single CRISPR RNA (crRNA) throughdirect RNA-DNA base-pairing to recognize a DNA target site that is inclose vicinity to a protospacer adjacent motif (PAM) (Jore, M. M. etal., 2011, Nat. Struct. Mol. Biol. 18:529-536, Westra, E. R. et al.,2012, Molecular Cell 46:595-605, and Sinkunas, T. et al., 2013, EMBO J.32:385-394).

Class I CRISPR-Cas Systems

Class I CRISPR-Cas systems comprise Types I, III, and IV. Acharacteristic feature of Class I systems is the presence of an effectorendonuclease complex instead of a single protein. A Cascade complexcomprises a RNA recognition motif (RRM) and a nucleic acid-bindingdomain that is the core fold of the diverse RAMP (Repeat-AssociatedMysterious Proteins) protein superfamily (Makarova et al. 2013, BiochemSoc Trans 41, 1392-1400; Makarova et al. 2015, Nature ReviewsMicrobiology Vol. 13:1-15). RAMP protein subunits include Cas5 and Cas7(which comprise the skeleton of the crRNA—effector complex), wherein theCas5 subunit binds the 5′ handle of the crRNA and interacts with thelarge subunit, and often includes Cas6 which is loosely associated withthe effector complex and typically functions as the repeat-specificRNase in the pre-crRNA processing (Charpentier et al., FEMS MicrobiolRev 2015, 39:428-441; Niewoehner et al., RNA 2016, 22:318-329).

Type I CRISPR-Cas systems comprise a complex of effector proteins,termed Cascade (CRISPR-associated complex for antiviral defense)comprising at a minimum Cas5 and Cas7. The effector complex functionstogether with a single CRISPR RNA (crRNA) and Cas3 to defend againstinvading viral DNA (Brouns, S. J. J. et al. Science 321:960-964;Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type ICRISPR-Cas loci comprise the signature gene cas3 (or a variant cas3′ orcas3″), which encodes a metal-dependent nuclease that possesses asingle-stranded DNA (ssDNA)-stimulated superfamily 2 helicase with ademonstrated capacity to unwind double stranded DNA (dsDNA) and RNA-DNAduplexes (Makarova et al. 2015, Nature Reviews; Microbiology Vol.13:1-15). Following target recognition, the Cas3 endonuclease isrecruited to the Cascade-crRNA-target DNA complex to cleave and degradethe DNA target (Westra, E. R. et al. (2012) Molecular Cell 46:595-605,Sinkunas, T. et al. (2011) EMBO J. 30:1335-1342, and Sinkunas, T. et al.(2013) EMBO J. 32:385-394). In some type I systems, Cas6 can be theactive endonuclease that is responsible for crRNA processing, and Cas5and Cas7 function as non-catalytic RNA-binding proteins; although intype I-C systems, crRNA processing can be catalyzed by Cas5 (Makarova etal. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type I systems aredivided into seven subtypes (Makarova et al. 2011, Nat Rev Microbiol.2011 9(6):467-477; Koonin et al. 2017, Curr Opinion Microbiology37:67-78). A modified type I CRISPR-associated complex for adaptiveantiviral defense (Cascade) comprising at least the protein subunitsCas7, Cas5 and Cas6, wherein one of these subunits is syntheticallyfused to a Cas3 endonuclease or a modified restriction endonuclease,FokI, have been described (WO2013098244 published 4 Jul. 4 2013).

Type III CRISPR-Cas systems, comprising a plurality of cas7 genes,target either ssRNA or ssDNA, and function as either an RNase as well asa target RNA-activated DNA nuclease (Tamulaitis et al., Trends inMicrobiology 25(10)49-61, 2017). Csm (Type III-A) and Cmr (Type III-B)complexes function as RNA-activated single-stranded (ss) DNases thatcouple the target RNA binding/cleavage with ssDNA degradation. Uponforeign DNA infection, the CRISPR RNA (crRNA)-guided binding of the Csmor Cmr complex to the emerging transcript recruits Cas10 DNase to theactively transcribed phage DNA, resulting in degradation of both thetranscript and phage DNA, but not the host DNA. The Cas10 HD-domain isresponsible for the ssDNase activity, and Csm3/Cmr4 subunits areresponsible for the endoribonuclease activity of the Csm/Cmr complex.The 3′-flanking sequence of the target RNA is critical for the ssDNaseactivity of Csm/Cmr: the basepairing with the 5′-handle of crRNAprotects host DNA from degradation.

Type IV systems, although comprising typical type I cas5 and cas7domains in addition to a cas8-like domain, may lack the CRISPR arraythat is characteristic of most other CRISPR-Cas systems.

Class II CRISPR-Cas Systems

Class II CRISPR-Cas systems comprise Types II, V, and VI. Acharacteristic feature of Class II systems is the presence of a singleCas effector protein instead of an effector complex. Types II and V Casproteins comprise an RuvC endonuclease domain that adopts the RNase Hfold.

Type II CRISPR/Cas systems employ a crRNA and tracrRNA (trans-activatingCRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNAcomprises a spacer region complementary to one strand of the doublestrand DNA target and a region that base pairs with the tracrRNA(trans-activating CRISPR RNA) forming a RNA duplex that directs the Casendonuclease to cleave the DNA target, leaving a blunt end. Spacers areacquired through a not fully understood process involving Cas1 and Cas2proteins. Type II CRISPR/Cas loci typically comprise cas1 and cas2 genesin addition to the cas9 gene (Chylinski et al., 2013, RNA Biology10:726-737; Makarova et al. 2015, Nature Reviews Microbiology Vol.13:1-15). Type II CRISR-Cas loci can encode a tracrRNA, which ispartially complementary to the repeats within the respective CRISPRarray, and can comprise other proteins such as Csn1 and Csn2. Thepresence of cas9 in the vicinity of cas1 and cas2 genes is the hallmarkof type II loci (Makarova et al. 2015, Nature Reviews Microbiology Vol.13:1-15).

Type V CRISPR/Cas systems comprise a single Cas endonuclease, includingCpf1 (Cas12) (Koonin et al., Curr Opinion Microbiology 37:67-78, 2017),that is an active RNA-guided endonuclease that does not necessarilyrequire the additional trans-activating CRISPR (tracr) RNA for targetcleavage, unlike Cas9.

Type VI CRISPR-Cas systems comprise a cas13 gene that encodes a nucleasewith two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding)domains but no HNH or RuvC domains, and are not dependent upon tracrRNAactivity. The majority of HEPN domains comprise conserved motifs thatconstitute a metal-independent endoRNase active site (Anantharam et al.,Biol Direct 8:15, 2013). Because of this feature, it is thought thattype VI systems act on RNA targets instead of the DNA targets that arecommon to other CRISPR-Cas systems.

Novel CRISPR-Cas Systems

Disclosed herein is a novel CRISPR-Cas system, components thereof, andmethods of using said components. The system comprises a novel Caseffector protein, Cas-alpha.

The novel CRISPR-Cas system components described herein may comprise oneor more subunits from different Cas systems, subunits derived ormodified from more than one different bacterial or archaeal prokaryote,and/or synthetic or engineered components.

Described herein is a newly identified CRISPR-Cas system comprisingnovel arrangements of cas genes. Further described are novel cas genesand proteins.

One feature of some of the novel Cas-alpha system is the locusarchitecture as depicted in FIGS. 1A-1D. In some aspects, the Cas-alphagenomic locus comprises a cas1 gene, a cas2 gene, a cas4 gene, and acas-alpha gene encoding the effector protein Cas-alpha. A CRISPR arraycomprising repeats of a nucleotide sequence may be found prior to, orfollowing, the gene encoding the Cas-alpha endonuclease. In someaspects, the cas-alpha locus may comprise a cas-alpha gene encoding aneffector protein, and a CRISPR array comprising repeats, but notcomprise any one or more of a cas1 gene, a cas2 gene, and/or a cas4gene.

CRISPR-Cas System Components Cas Proteins

A number of proteins may be encoded in the CRISPR cas operon, includingthose involved in adaptation (spacer insertion), interference (effectormodule target binding, target nicking or cleavage—e.g. endonucleaseactivity), expression (pre-crRNA processing), regulation, or other.

Two proteins, Cas1 and Cas2, are conserved among many CRISPR systems(for example, as described in Koonin et al., Curr Opinion Microbiology37:67-78, 2017). Cas1 is a metal-dependent DNA-specific endonucleasethat produces double-stranded DNA fragments. In some systems Cas1 formsa stable complex with Cas2, which is essential to spacer acquisition andinsertion for CRISPR systems (Nuñez et al., Nature Str Mol Biol21:528-534, 2014).

A number of other proteins have been identified across differentsystems, including Cas4 (which may have similarity to a RecB nuclease)and is thought to play a role in the capture of new viral DNA sequencesfor incorporation into the CRISPR array (Zhang et al., PLOS One7(10):e47232, 2012).

Some proteins may encompass a plurality of functions. For example, Cas9,the signature protein of Class 2 type II systems, has been demonstratedto be involved in pre-crRNA processing, target binding, as well astarget cleavage.

The novel Cas-alpha proteins disclosed herein include effector proteins(endonucleases) as well as adaptation proteins. Cas endonucleases havebeen identified from several bacterial and archaebacterial sources, andinclude those presented in FIGS. 7A-7K.

Cas Endonucleases and Effectors

Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain, and include restriction endonucleases that cleaveDNA at specific sites without damaging the bases. Examples ofendonucleases include restriction endonucleases, meganucleases, TALeffector nucleases (TALENs), zinc finger nucleases, and Cas(CRISPR-associated) effector endonucleases.

Cas endonucleases, either as single effector proteins or in an effectorcomplex with other components, unwind the DNA duplex at the targetsequence and optionally cleave at least one DNA strand, as mediated byrecognition of the target sequence by a polynucleotide (such as, but notlimited to, a crRNA or guide RNA) that is in complex with the Caseffector protein. Such recognition and cutting of a target sequence by aCas endonuclease typically occurs if the correct protospacer-adjacentmotif (PAM) is located at or adjacent to the 3′ end of the DNA targetsequence. Alternatively, a Cas endonuclease herein may lack DNA cleavageor nicking activity, but can still specifically bind to a DNA targetsequence when complexed with a suitable RNA component. (See also U.S.Patent Application US20150082478 published 19 Mar. 2015 andUS20150059010 published 26 Feb. 2015).

Cas endonucleases may occur as individual effectors (Class 2 CRISPRsystems) or as part of larger effector complexes (Class I CRISPRsystems).

Cas endonucleases that have been described include, but are not limitedto, for example:Cas3 (a feature of Class 1 type I systems), Cas9 (afeature of Class 2 type II systems) and Cas12 (Cpf1) (a feature of Class2 type V systems). Cas3 (and its variants Cas3′ and Cas3″) functions asa single-stranded DNA nuclease (HD domain) and an ATP-dependenthelicase. A variant of the Cas3 endonuclease can be obtained bydisabling the functional activity of one or both domains of the Cas3endonuclease poly peptide. Disabling the ATPase dependent helicaseactivity (by deletion, knockout of the Cas3-helicase domain, or throughmutagenesis of critical residues or by assembling the reaction in theabsence of ATP as described previously (Sinkunas, T. et al., 2013, EMBOJ. 32:385-394) can convert the cleavage ready Cascade comprising themodified Cas3 endonuclease into a nickase (as the HD domain is stillfunctional). Disabling the HD endonuclease activity can be accomplishedby any method known in the art, such as but not limited to, mutagenesisof critical residues of the HD domain, can convert the cleavage readyCascade comprising the modified Cas3 endonuclease into a helicase.Disabling the both the Cas helicase and Cas3 HD endonuclease activitycan be accomplished by any method known in the art, such as but notlimited to, mutagenesis of critical residues of both the helicase and HDdomains, can convert the cleavage ready Cascade comprising the modifiedCas3 endonuclease into a binder protein that binds to a target sequence.

Cas9 (formerly referred to as Cas5, Csn1, or Csx12) is a Casendonuclease that forms a complex with a crNucleotide and atracrNucleotide, or with a single guide polynucleotide, for specificallyrecognizing and cleaving all or part of a DNA target sequence. Cas9recognizes a 3′ GC-rich PAM sequence on the target dsDNA. A Cas9 proteincomprises a RuvC nuclease with an HNH (H—N—H) nuclease adjacent to theRuvC-II domain. The RuvC nuclease and HNH nuclease each can cleave asingle DNA strand at a target sequence (the concerted action of bothdomains leads to DNA double-strand cleavage, whereas activity of onedomain leads to a nick). In general, the RuvC domain comprisessubdomains I, II and III, where domain I is located near the N-terminusof Cas9 and subdomains II and III are located in the middle of theprotein, flanking the HNH domain (Hsu et al., 2013, Cell 157:1262-1278).Cas9 endonucleases are typically derived from a type II CRISPR system,which includes a DNA cleavage system utilizing a Cas9 endonuclease incomplex with at least one polynucleotide component. For example, a Cas9can be in complex with a CRISPR RNA (crRNA) and a trans-activatingCRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex witha single guide RNA (Makarova et al. 2015, Nature Reviews MicrobiologyVol. 13:1-15).

Cas12 (formerly referred to as Cpf1, and variants c2c1, c2c3, CasX, andCasY) comprise an RuvC nuclease domain and produced staggered, 5′overhangs on the dsDNA target. Some variants do not require a tracrRNA,unlike the functionality of Cas9. Cas12 and its variants recognize a 5′AT-rich PAM sequence on the target dsDNA. An insert domain, called Nuc,of the Cas12a protein has been demonstrated to be responsible for targetstrand cleavage (Yamano et al., Cell 2016, 165:949-962). Additionalmutation studies in other Cas12 proteins demonstrated the Nuc domaincontributes to guide and target binding, with the RuvC domainresponsible for cleavage (Swarts et al., Mol Cell 2017, 66:221-233e224).

Cas endonucleases and effector proteins can be used for targeted genomeediting (via simplex and multiplex double-strand breaks and nicks) andtargeted genome regulation (via tethering of epigenetic effector domainsto either the Cas protein or sgRNA. A Cas endonuclease can also beengineered to function as an RNA-guided recombinase, and via RNA tetherscould serve as a scaffold for the assembly of multiprotein and nucleicacid complexes (Mali et al., 2013, Nature Methods Vol. 10:957-963).

Cas-alpha Endonucleases

A Cas-alpha endonuclease is defined as a functional RNA-guided,PAM-dependent dsDNA cleavage protein of fewer than 800 amino acids,comprising: a C-terminal RuvC catalytic domain split into threesubdomains and further comprising bridge-helix and one or more Zincfinger motif(s); and an N-terminal Rec subunit with a helical bundle,WED wedge-like (or “Oligonucleotide Binding Domain”, OBD) domain, and,optionally, a Zinc finger motif.

A Cas-alpha endonuclease comprises, when aligned to SEQ ID NO:17,relative to the amino acid position numbers of SEQ ID NO:17, at leastone, at least two, at least three, at least four, at least five, atleast six, or seven of the following: a Glycine (G) at position 337, aGlycine (G) at position 341, a Glutamic Acid (E) at position 430, aLeucine (L) at position 432, a Cysteine (C) at position 487, a Cysteine(C) at position 490, a Cysteine (C) at position 507, and/or a Cysteine(C) or Histidine (H) at position 512. A Cas-alpha endonucleasecomprises, the following motifs: GxxxG, ExL, Cx_(n)C, Cx_(n)(C or H)(where n=one or more amino acids).

RuvC domains have been demonstrated in the literature to encompassendonuclease functionality. A Cas-alpha endonuclease may be isolated oridentified from a locus that comprises a cas-alpha gene encoding aneffector protein, and an array comprising a plurality repeats. In someaspects, a cas-alpha locus may further comprise a partial or whole cas1gene, a cas2 gene, and/or a cas4 gene.

Zinc finger motifs are domains that coordinate one or more zinc ions,usually through Cysteine and Histidine sidechains, to stabilize theirfold. Zinc fingers are named for the pattern of Cysteine and Histidineresidues that coordinate the zinc ion (e.g., C4 means a zinc ion iscoordinated by four Cysteine residues; C3H means a zinc ion iscoordinated by three Cysteine residues and one Histidine residue).

Cas-alpha proteins comprise one or more Zinc Finger (ZFN) coordinationmotif(s) that may form a Zinc binding domain. Zinc Finger-like motifscan aid in target and non-target strand separation and loading of theguide RNA into the DNA target. Cas-alpha proteins comprising one or moreZinc Finger motifs may provide additional stability to theribonucleoprotein complex on the target polynucleotide. Cas-alphaproteins comprise C4 or C3H zinc binding domains.

Some Cas-alpha proteins and polynucleotides are given in FIGS. 7A-7K,with key structural motifs of the endonuclease proteins depicted inFIGS. 8A-8K, respectively.

Cas-alpha endonucleases are RNA-guided endonucleases capable of bindingto, and cleaving, a double-strand DNA target that comprises: (1) asequence sharing homology with a nucleotide sequence of the guide RNA,and (2) a PAM sequence. In some aspects, the PAM is T-rich. In someaspects, the PAM is C-rich.

A Cas-alpha endonuclease is functional as a double-strand-break-inducingagent, and may also be a nickase, or a single-strand-break inducingagent. In some aspects, a catalytically inactive Cas-alpha endonucleasemay be used to target or recruit to a target DNA sequence but not inducecleavage. In some aspects, a catalytically inactive Cas-alpha proteinmay be used with a functional endonuclease, to cleave a target sequence.In some aspects, a catalytically inactive Cas-alpha protein may becombined with a base editing molecule, such as a deaminase. In someaspects, a deaminase may be a cytidine deaminase. In some aspects, adeaminase may be an adenine deaminase. In some aspects, a deaminase maybe ADAR-2.

A Cas-alpha endonuclease is further defined as an RNA-guideddouble-strand DNA cleavage protein that shares at least 50%, between 50%and 55%, at least 55%, between 55% and 60%, at least 60%, between 60%and 65%, at least 65%, between 65% and 70%, at least 70%, between 70%and 75%, at least 75%, between 75% and 80%, at least 80%, between 80%and 85%, at least 85%, between 85% and 90%, at least 90%, between 90%and 95%, at least 95%, between 95% and 96%, at least 96%, between 96%and 97%, at least 97%, between 97% and 98%, at least 98%, between 98%and 99%, at least 99%, between 99% and 100%, or 100% sequence identitywith at least 50, between 50 and 100, at least 100, between 100 and 150,at least 150, between 150 and 200, at least 200, between 200 and 250, atleast 250, between 250 and 300, at least 300, between 300 and 350, atleast 350, between 350 and 400, at least 400, between 400 and 450, atleast 500, or greater than 500 contiguous amino acids of any of SEQ IDNOs: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, and 371, or a functional fragment thereof, or functional variantthereof that retains at least partial activity. A “functional fragment”of a Cas-alpha endonuclease retains the ability to recognize, or bind,or nick a single strand of a double-stranded polynucleotide, or cleaveboth strands of a double-stranded polynucleotide, or any combination ofthe preceding.

The Cas-alpha endonuclease may be encoded by a polynucleotide thatshares at least 50%, between 50% and 55%, at least 55%, between 55% and60%, at least 60%, between 60% and 65%, at least 65%, between 65% and70%, at least 70%, between 70% and 75%, at least 75%, between 75% and80%, at least 80%, between 80% and 85%, at least 85%, between 85% and90%, at least 90%, between 90% and 95%, at least 95%, between 95% and96%, at least 96%, between 96% and 97%, at least 97%, between 97% and98%, at least 98%, between 98% and 99%, at least 99%, between 99% and100%, or 100% sequence identity with at least 50, between 50 and 100, atleast 100, between 100 and 150, at least 150, between 150 and 200, atleast 200, between 200 and 250, at least 250, between 250 and 300, atleast 300, between 300 and 350, at least 350, between 350 and 400, atleast 400, between 400 and 450, at least 500, between 500 and 550, atleast 600, between 600 and 650, at least 650, between 650 and 700, atleast 700, between 700 and 750, at least 750, between 750 and 800, atleast 800, between 800 and 850, at least 850, between 850 and 900, atleast 900, between 900 and 950, at least 950, between 950 and 1000, atleast 1000, or even greater than 1000 contiguous nucleotides of any ofSEQ ID NOs: 13, 14, 15, 16, 25, 26, 27, 28, 29, 30, or 31, or encodesany one of SEQ ID NOs: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, and 371.

A Cas endonuclease, effector protein, or functional fragment thereof,for use in the disclosed methods, can be isolated from a native source,or from, a recombinant source where the genetically modified host cellis modified to express the nucleic acid sequence encoding the protein.Alternatively, the Cas protein can be produced using cell free proteinexpression systems, or be synthetically produced. Effector Cas nucleasesmay be isolated and introduced into a heterologous cell, or may bemodified from its native form to exhibit a different type or magnitudeof activity than what it would exhibit in its native source. Suchmodifications include but are not limited to: fragments, variants,substitutions, deletions, and insertions.

Fragments and variants of Cas endonucleases and Cas effector proteinscan be obtained via methods such as site-directed mutagenesis andsynthetic construction. Methods for measuring endonuclease activity arewell known in the art such as, but not limiting to, WO2013166113published 7 Nov. 2013, WO2016186953 published 24 Nov. 2016, andWO2016186946 published 24 Nov. 2016.

The Cas endonuclease can comprise a modified form of the Caspolypeptide. The modified form of the Cas polypeptide can include anamino acid change (e.g., deletion, insertion, or substitution) thatreduces the naturally-occurring nuclease activity of the Cas protein.For example, in some instances, the modified form of the Cas protein hasless than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1% of the nuclease activity of thecorresponding wild-type Cas polypeptide (US20140068797 published 6 Mar.2014). In some cases, the modified form of the Cas polypeptide has nosubstantial nuclease activity and is referred to as catalytically“inactivated Cas” or “deactivated Cas (dCas).” An inactivatedCas/deactivated Cas includes a deactivated Cas endonuclease (dCas). Acatalytically inactive Cas effector protein can be fused to aheterologous sequence to induce or modify activity.

A Cas endonuclease can be part of a fusion protein comprising one ormore heterologous protein domains (e.g., 1, 2, 3, or more domains inaddition to the Cas protein). Such a fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains, such as between Cas and a first heterologous domain.Examples of protein domains that may be fused to a Cas protein hereininclude, without limitation, epitope tags (e.g., histidine [His], V5,FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]),reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase[HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase,beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP],HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein[YFP], blue fluorescent protein [BFP]), and domains having one or moreof the following activities: methylase activity, demethylase activity,transcription activation activity (e.g., VP16 or VP64), transcriptionrepression activity, transcription release factor activity, histonemodification activity, RNA cleavage activity and nucleic acid bindingactivity. A Cas protein can also be in fusion with a protein that bindsDNA molecules or other molecules, such as maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, andherpes simplex virus (HSV) VP16.

A catalytically active and/or inactive Cas endonuclease can be fused toa heterologous sequence (US20140068797 published 6 Mar. 2014). Suitablefusion partners include, but are not limited to, a polypeptide thatprovides an activity that indirectly increases transcription by actingdirectly on the target DNA or on a polypeptide (e.g., a histone or otherDNA-binding protein) associated with the target DNA. Additional suitablefusion partners include, but are not limited to, a polypeptide thatprovides for methyltransferase activity, demethylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity, or demyristoylation activity. Furthersuitable fusion partners include, but are not limited to, a polypeptidethat directly provides for increased transcription of the target nucleicacid (e.g., a transcription activator or a fragment thereof, a proteinor fragment thereof that recruits a transcription activator, a smallmolecule/drug-responsive transcription regulator, etc.). A partiallyactive or catalytically inactive Cas-alpha endonuclease can also befused to another protein or domain, for example Clo51 or FokI nuclease,to generate double-strand breaks (Guilinger et al. Nature Biotechnology,volume 32, number 6, June 2014).

A catalytically active or inactive Cas protein, such as the Cas-alphaprotein described herein, can also be in fusion with a molecule thatdirects editing of single or multiple bases in a polynucleotidesequence, for example a site-specific deaminase that can change theidentity of a nucleotide, for example from C•G to T•A or an A•T to G•C(Gaudelli et al., Programmable base editing of A•T to G•C in genomic DNAwithout DNA cleavage.” Nature (2017); Nishida et al. “Targetednucleotide editing using hybrid prokaryotic and vertebrate adaptiveimmune systems.” Science 353 (6305) (2016); Komor et al. “Programmableediting of a target base in genomic DNA without double-stranded DNAcleavage.” Nature 533 (7603) (2016):420-4. A base editing fusion proteinmay comprise, for example, an active (double strand break creating),partially active (nickase) or deactivated (catalytically inactive)Cas-alpha endonuclease and a deaminase (such as, but not limited to, acytidine deaminase, an adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3,BE4, ABEs, or the like). Base edit repair inhibitors and glycosylaseinhibitors (e.g., uracil glycosylase inhibitor (to prevent uracilremoval)) are contemplated as other components of a base editing system,in some embodiments.

The Cas endonucleases described herein can be expressed and purified bymethods known in the art, for example as described in WO/2016/186953published 24 Nov. 2016.

Many Cas endonucleases have been described to date that can recognizespecific PAM sequences (WO2016186953 published 24 Nov. 2016,WO2016186946 published 24 Nov. 2016, and Zetsche B et al. 2015. Cell163, 1013) and cleave the target DNA at a specific position. It isunderstood that based on the methods and embodiments described hereinutilizing a novel guided Cas system one skilled in the art can nowtailor these methods such that they can utilize any guided endonucleasesystem.

A Cas effector protein can comprise a heterologous nuclear localizationsequence (NLS). A heterologous NLS amino acid sequence herein may be ofsufficient strength to drive accumulation of a Cas protein in adetectable amount in the nucleus of a yeast cell herein, for example. AnNLS may comprise one (monopartite) or more (e.g., bipartite) shortsequences (e.g., 2 to 20 residues) of basic, positively charged residues(e.g., lysine and/or arginine), and can be located anywhere in a Casamino acid sequence but such that it is exposed on the protein surface.An NLS may be operably linked to the N-terminus or C-terminus of a Casprotein herein, for example. Two or more NLS sequences can be linked toa Cas protein, for example, such as on both the N- and C-termini of aCas protein. The Cas endonuclease gene can be operably linked to a SV40nuclear targeting signal upstream of the Cas codon region and abipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc.Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region.Non-limiting examples of suitable NLS sequences herein include thosedisclosed in U.S. Pat. Nos. 6,660,830 and 7,309,576.

Guide Polynucleotides

The guide polynucleotide enables target recognition, binding, andoptionally cleavage by the Cas endonuclease, and can be a singlemolecule or a double molecule. The guide polynucleotide sequence can bea RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence). Optionally, the guide polynucleotide can compriseat least one nucleotide, phosphodiester bond or linkage modificationsuch as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC,2,6-Diaminopurine, 2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA,phosphorothioate bond, linkage to a cholesterol molecule, linkage to apolyethylene glycol molecule, linkage to a spacer 18 (hexaethyleneglycol chain) molecule, or 5′ to 3′ covalent linkage resulting incircularization. A guide polynucleotide that solely comprisesribonucleic acids is also referred to as a “guide RNA” or “gRNA”(US20150082478 published 19 Mar. 2015 and US20150059010 published 26Feb. 2015). A guide polynucleotide may be engineered or synthetic.

The guide polynucleotide includes a chimeric non-naturally occurringguide RNA comprising regions that are not found together in nature(i.e., they are heterologous with each other). For example, a chimericnon-naturally occurring guide RNA comprising a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that canhybridize to a nucleotide sequence in a target DNA, linked to a secondnucleotide sequence that can recognize the Cas endonuclease, such thatthe first and second nucleotide sequence are not found linked togetherin nature.

The guide polynucleotide can be a double molecule (also referred to asduplex guide polynucleotide) comprising a crNucleotide sequence (such asa crRNA) and a tracrNucleotide (such as a tracrRNA) sequence. In somecases, there is a linker polynucleotide that connects the crRNA andtracrRNA to form a single guide, for example an sgRNA.

The crNucleotide includes a first nucleotide sequence domain (referredto as Variable Targeting domain or VT domain) that can hybridize to anucleotide sequence in a target DNA and a second nucleotide sequence(also referred to as a tracr mate sequence) that is part of a Casendonuclease recognition (CER) domain. The tracr mate sequence canhybridized to a tracrNucleotide along a region of complementarity andtogether form the Cas endonuclease recognition domain or CER domain. TheCER domain is capable of interacting with a Cas endonucleasepolypeptide. The crNucleotide and the tracrNucleotide of the duplexguide polynucleotide can be RNA, DNA, and/or RNA-DNA-combinationsequences. In some embodiments, the crNucleotide molecule of the duplexguide polynucleotide is referred to as “crDNA” (when composed of acontiguous stretch of DNA nucleotides) or “crRNA” (when composed of acontiguous stretch of RNA nucleotides), or “crDNA-RNA” (when composed ofa combination of DNA and RNA nucleotides). The crNucleotide can comprisea fragment of the crRNA naturally occurring in Bacteria and Archaea. Thesize of the fragment of the crRNA naturally occurring in Bacteria andArchaea that can be present in a crNucleotide disclosed herein can rangefrom, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, a crRNAmolecule is selected from the group consisting of: SEQ ID NOs: 57, 58,and 59.

In some embodiments the tracrNucleotide is referred to as “tracrRNA”(when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA”(when composed of a contiguous stretch of DNA nucleotides) or“tracrDNA-RNA” (when composed of a combination of DNA and RNAnucleotides. In one embodiment, the RNA that guides the RNA/Cas9endonuclease complex is a duplexed RNA comprising a duplexcrRNA-tracrRNA. The tracrRNA (trans-activating CRISPR RNA) comprises, inthe 5′-to-3′ direction, (i) a sequence that anneals with the repeatregion of CRISPR type II crRNA and (ii) a stem loop-comprising portion(Deltcheva et al., Nature 471:602-607). The duplex guide polynucleotidecan form a complex with a Cas endonuclease, wherein said guidepolynucleotide/Cas endonuclease complex (also referred to as a guidepolynucleotide/Cas endonuclease system) can direct the Cas endonucleaseto a genomic target site, enabling the Cas endonuclease to recognize,bind to, and optionally nick or cleave (introduce a single ordouble-strand break) into the target site. (US20150082478 published 19Mar. 2015 and US20150059010 published 26 Feb. 2015).

In some embodiments, a tracrRNA molecule is selected from the groupconsisting of: SEQ ID NOs: 60-68.

In one aspect, the guide polynucleotide is a guide polynucleotidecapable of forming a PGEN as described herein, wherein said guidepolynucleotide comprises a first nucleotide sequence domain that iscomplementary to a nucleotide sequence in a target DNA, and a secondnucleotide sequence domain that interacts with said Cas endonucleasepolypeptide.

In one aspect, the guide polynucleotide is a guide polynucleotidedescribed herein, wherein the first nucleotide sequence and the secondnucleotide sequence domain is selected from the group consisting of aDNA sequence, a RNA sequence, and a combination thereof.

In one aspect, the guide polynucleotide is a guide polynucleotidedescribed herein, wherein the first nucleotide sequence and the secondnucleotide sequence domain is selected from the group consisting of RNAbackbone modifications that enhance stability, DNA backbonemodifications that enhance stability, and a combination thereof (seeKanasty et al., 2013, Common RNA-backbone modifications, NatureMaterials 12:976-977; US20150082478 published 19 Mar. 2015 andUS20150059010 published 26 Feb. 2015)

The guide RNA includes a dual molecule comprising a chimericnon-naturally occurring crRNA linked to at least one tracrRNA. Achimeric non-naturally occurring crRNA includes a crRNA that comprisesregions that are not found together in nature (i.e., they areheterologous with each other. For example, a crRNA comprising a firstnucleotide sequence domain (referred to as Variable Targeting domain orVT domain) that can hybridize to a nucleotide sequence in a target DNA,linked to a second nucleotide sequence (also referred to as a tracr matesequence) such that the first and second sequence are not found linkedtogether in nature.

The guide polynucleotide can also be a single molecule (also referred toas single guide polynucleotide) comprising a crNucleotide sequencelinked to a tracrNucleotide sequence. The single guide polynucleotidecomprises a first nucleotide sequence domain (referred to as VariableTargeting domain or VT domain) that can hybridize to a nucleotidesequence in a target DNA and a Cas endonuclease recognition domain (CERdomain), that interacts with a Cas endonuclease polypeptide. In someembodiments, an sgRNA molecule is selected from the group consisting of:SEQ ID NOs: 69-77.

The VT domain and/or the CER domain of a single guide polynucleotide cancomprise a RNA sequence, a DNA sequence, or a RNA-DNA-combinationsequence. The single guide polynucleotide being comprised of sequencesfrom the crNucleotide and the tracrNucleotide may be referred to as“single guide RNA” (when composed of a contiguous stretch of RNAnucleotides) or “single guide DNA” (when composed of a contiguousstretch of DNA nucleotides) or “single guide RNA-DNA” (when composed ofa combination of RNA and DNA nucleotides). The single guidepolynucleotide can form a complex with a Cas endonuclease, wherein saidguide polynucleotide/Cas endonuclease complex (also referred to as aguide polynucleotide/Cas endonuclease system) can direct the Casendonuclease to a genomic target site, enabling the Cas endonuclease torecognize, bind to, and optionally nick or cleave (introduce a single ordouble-strand break) the target site. (US20150082478 published 19 Mar.2015 and US20150059010 published 26 Feb. 2015).

A chimeric non-naturally occurring single guide RNA (sgRNA) includes asgRNA that comprises regions that are not found together in nature(i.e., they are heterologous with each other. For example, a sgRNAcomprising a first nucleotide sequence domain (referred to as VariableTargeting domain or VT domain) that can hybridize to a nucleotidesequence in a target DNA linked to a second nucleotide sequence (alsoreferred to as a tracr mate sequence) that are not found linked togetherin nature.

The nucleotide sequence linking the crNucleotide and the tracrNucleotideof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA combination sequence. In one embodiment, thenucleotide sequence linking the crNucleotide and the tracrNucleotide ofa single guide polynucleotide (also referred to as “loop”) can be atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length. In anotherembodiment, the nucleotide sequence linking the crNucleotide and thetracrNucleotide of a single guide polynucleotide can comprise atetraloop sequence, such as, but not limiting to a GAAA tetraloopsequence.

The guide polynucleotide can be produced by any method known in the art,including chemically synthesizing guide polynucleotides (such as but notlimiting to Hendel et al. 2015, Nature Biotechnology 33, 985-989), invitro generated guide polynucleotides, and/or self-splicing guide RNAs(such as but not limited to Xie et al. 2015, PNAS 112:3570-3575).

Protospacer Adjacent Motif (PAM)

A “protospacer adjacent motif” (PAM) herein refers to a short nucleotidesequence adjacent to a target sequence (protospacer) that can berecognized (targeted) by a guide polynucleotide/Cas endonuclease system.The Cas endonuclease may not successfully recognize a target DNAsequence if the target DNA sequence is not followed by a PAM sequence.The sequence and length of a PAM herein can differ depending on the Casprotein or Cas protein complex used. The PAM sequence can be of anylength but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 nucleotides long.

A “randomized PAM” and “randomized protospacer adjacent motif” are usedinterchangeably herein, and refer to a random DNA sequence adjacent to atarget sequence (protospacer) that is recognized (targeted) by a guidepolynucleotide/Cas endonuclease system. The randomized PAM sequence canbe of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long. A randomizednucleotide includes anyone of the nucleotides A, C, G or T.

Guide Polynucleotide/Cas Endonuclease Complexes

A guide polynucleotide/Cas endonuclease complex described herein iscapable of recognizing, binding to, and optionally nicking, unwinding,or cleaving all or part of a target sequence.

A guide polynucleotide/Cas endonuclease complex that can cleave bothstrands of a DNA target sequence typically comprises a Cas protein thathas all of its endonuclease domains in a functional state (e.g., wildtype endonuclease domains or variants thereof retaining some or allactivity in each endonuclease domain). Thus, a wild type Cas protein(e.g., a Cas protein disclosed herein), or a variant thereof retainingsome or all activity in each endonuclease domain of the Cas protein, isa suitable example of a Cas endonuclease that can cleave both strands ofa DNA target sequence.

A guide polynucleotide/Cas endonuclease complex that can cleave onestrand of a DNA target sequence can be characterized herein as havingnickase activity (e.g., partial cleaving capability). A Cas nickasetypically comprises one functional endonuclease domain that allows theCas to cleave only one strand (i.e., make a nick) of a DNA targetsequence. For example, a Cas9 nickase may comprise (i) a mutant,dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wildtype HNH domain). As another example, a Cas9 nickase may comprise (i) afunctional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant,dysfunctional HNH domain. Non-limiting examples of Cas9 nickasessuitable for use herein are disclosed in US20140189896 published on 3Jul. 2014. A pair of Cas nickases can be used to increase thespecificity of DNA targeting. In general, this can be done by providingtwo Cas nickases that, by virtue of being associated with RNA componentswith different guide sequences, target and nick nearby DNA sequences onopposite strands in the region for desired targeting. Such nearbycleavage of each DNA strand creates a double-strand break (i.e., a DSBwith single-stranded overhangs), which is then recognized as a substratefor non-homologous-end-joining, NHEJ (prone to imperfect repair leadingto mutations) or homologous recombination, HR. Each nick in theseembodiments can be at least about 5, between 5 and 10, at least 10,between 10 and 15, at least 15, between 15 and 20, at least 20, between20 and 30, at least 30, between 30 and 40, at least 40, between 40 and50, at least 50, between 50 and 60, at least 60, between 60 and 70, atleast 70, between 70 and 80, at least 80, between 80 and 90, at least90, between 90 and 100, or 100 or greater (or any integer between 5 and100) bases apart from each other, for example. One or two Cas nickaseproteins herein can be used in a Cas nickase pair. For example, a Cas9nickase with a mutant RuvC domain, but functioning HNH domain (i.e.,Cas9 HNH+/RuvC−), can be used (e.g., Streptococcus pyogenes Cas9HNH+/RuvC−). Each Cas9 nickase (e.g., Cas9 HNH+/RuvC−) can be directedto specific DNA sites nearby each other (up to 100 base pairs apart) byusing suitable RNA components herein with guide RNA sequences targetingeach nickase to each specific DNA site.

A guide polynucleotide/Cas endonuclease complex in certain embodimentscan bind to a DNA target site sequence, but does not cleave any strandat the target site sequence. Such a complex may comprise a Cas proteinin which all of its nuclease domains are mutant, dysfunctional. Forexample, a Cas9 protein that can bind to a DNA target site sequence, butdoes not cleave any strand at the target site sequence, may compriseboth a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNHdomain. A Cas protein herein that binds, but does not cleave, a targetDNA sequence can be used to modulate gene expression, for example, inwhich case the Cas protein could be fused with a transcription factor(or portion thereof) (e.g., a repressor or activator, such as any ofthose disclosed herein).

In one aspect, the guide polynucleotide/Cas endonuclease complex (PGEN)described herein is a PGEN, wherein said Cas endonuclease is optionallycovalently or non-covalently linked, or assembled to at least oneprotein subunit, or functional fragment thereof.

In one embodiment of the disclosure, the guide polynucleotide/Casendonuclease complex is a guide polynucleotide/Cas endonuclease complex(PGEN) comprising at least one guide polynucleotide and at least one Casendonuclease polypeptide, wherein said Cas endonuclease polypeptidecomprises at least one protein subunit, or a functional fragmentthereof, wherein said guide polynucleotide is a chimeric non-naturallyoccurring guide polynucleotide, wherein said guide polynucleotide/Casendonuclease complex is capable of recognizing, binding to, andoptionally nicking, unwinding, or cleaving all or part of a targetsequence.

The Cas effector protein can be a Cas-alpha effector protein asdisclosed herein.

In one embodiment of the disclosure, the guide polynucleotide/Caseffector complex is a guide polynucleotide/Cas effector protein complex(PGEN) comprising at least one guide polynucleotide and a Cas-alphaeffector protein, wherein said guide polynucleotide/Cas effector proteincomplex is capable of recognizing, binding to, and optionally nicking,unwinding, or cleaving all or part of a target sequence.

The PGEN can be a guide polynucleotide/Cas effector protein complex,wherein said Cas effector protein further comprises one copy or multiplecopies of at least one protein subunit, or a functional fragmentthereof. In some embodiments, said protein subunit is selected from thegroup consisting of a Cas1 protein subunit, a Cas2 protein subunit, aCas4 protein subunit, and any combination thereof. The PGEN can be aguide polynucleotide/Cas effector protein complex, wherein said Caseffector protein further comprises at least two different proteinsubunits of selected from the group consisting of a Cas1, Cas2, andCas4.

The PGEN can be a guide polynucleotide/Cas effector protein complex,wherein said Cas effector protein further comprises at least threedifferent protein subunits, or functional fragments thereof, selectedfrom the group consisting of Cas1, Cas2, and one additional Cas protein,optionally comprising Cas4.

In one aspect, the guide polynucleotide/Cas effector protein complex(PGEN) described herein is a PGEN, wherein said Cas effector protein iscovalently or non-covalently linked to at least one protein subunit, orfunctional fragment thereof. The PGEN can be a guide polynucleotide/Caseffector protein complex, wherein said Cas effector protein polypeptideis covalently or non-covalently linked, or assembled to one copy ormultiple copies of at least one protein subunit, or a functionalfragment thereof, selected from the group consisting of a Cas1 proteinsubunit, a Cas2 protein subunit, a one additional Cas protein optionallycomprising Cas4 protein subunit, and any combination thereof. The PGENcan be a guide polynucleotide/Cas effector protein complex, wherein saidCas effector protein is covalently or non-covalently linked or assembledto at least two different protein subunits selected from the groupconsisting of a Cas1, a Cas2, and one additional Cas protein, optionallycomprising Cas4. The PGEN can be a guide polynucleotide/Cas effectorprotein complex, wherein said Cas effector protein is covalently ornon-covalently linked to at least three different protein subunits, orfunctional fragments thereof, selected from the group consisting of aCas1, a Cas2, and one additional Cas protein, optionally comprisingCas4, and any combination thereof.

Any component of the guide polynucleotide/Cas effector protein complex,the guide polynucleotide/Cas effector protein complex itself, as well asthe polynucleotide modification template(s) and/or donor DNA(s), can beintroduced into a heterologous cell or organism by any method known inthe art.

Recombinant Constructs for Transformation of Cells

The disclosed guide polynucleotides, Cas endonucleases, polynucleotidemodification templates, donor DNAs, guide polynucleotide/Casendonuclease systems disclosed herein, and any one combination thereof,optionally further comprising one or more polynucleotide(s) of interest,can be introduced into a cell. Cells include, but are not limited to,human, non-human, animal, bacterial, fungal, insect, yeast,non-conventional yeast, and plant cells as well as plants and seedsproduced by the methods described herein.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal., Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y. (1989). Transformation methods arewell known to those skilled in the art and are described infra.

Vectors and constructs include circular plasmids, and linearpolynucleotides, comprising a polynucleotide of interest and optionallyother components including linkers, adapters, regulatory or analysis. Insome examples a recognition site and/or target site can be comprisedwithin an intron, coding sequence, 5′ UTRs, 3′ UTRs, and/or regulatoryregions.

Components for Expression and Utilization of Novel CRISPR-Cas Systems inProkaryotic and Eukaryotic Cells

The invention further provides expression constructs for expressing in aprokaryotic or eukaryotic cell/organism a guide RNA/Cas system that iscapable of recognizing, binding to, and optionally nicking, unwinding,or cleaving all or part of a target sequence.

In one embodiment, the expression constructs of the disclosure comprisea promoter operably linked to a nucleotide sequence encoding a Cas gene(or plant optimized, including a Cas endonuclease gene described herein)and a promoter operably linked to a guide RNA of the present disclosure.The promoter is capable of driving expression of an operably linkednucleotide sequence in a prokaryotic or eukaryotic cell/organism.

Nucleotide sequence modification of the guide polynucleotide, VT domainand/or CER domain can be selected from, but not limited to, the groupconsisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence,a stability control sequence, a sequence that forms a dsRNA duplex, amodification or sequence that targets the guide poly nucleotide to asubcellular location, a modification or sequence that provides fortracking, a modification or sequence that provides a binding site forproteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro Unucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage,or any combination thereof. These modifications can result in at leastone additional beneficial feature, wherein the additional beneficialfeature is selected from the group of a modified or regulated stability,a subcellular targeting, tracking, a fluorescent label, a binding sitefor a protein or protein complex, modified binding affinity tocomplementary target sequence, modified resistance to cellulardegradation, and increased cellular permeability.

A method of expressing RNA components such as gRNA in eukaryotic cellsfor performing Cas9-mediated DNA targeting has been to use RNApolymerase III (Pol III) promoters, which allow for transcription of RNAwith precisely defined, unmodified, 5′- and 3′-ends (DiCarlo et al.,Nucleic Acids Res. 41:4336-4343; Ma et al., Mol. Ther. Nucleic Acids3:e161). This strategy has been successfully applied in cells of severaldifferent species including maize and soybean (US20150082478 published19 Mar. 2015). Methods for expressing RNA components that do not have a5′ cap have been described (WO2016/025131 published 18 Feb. 2016).

Various methods and compositions can be employed to obtain a cell ororganism having a polynucleotide of interest inserted in a target sitefor a Cas endonuclease. Such methods can employ homologous recombination(HR) to provide integration of the polynucleotide of interest at thetarget site. In one method described herein, a polynucleotide ofinterest is introduced into the organism cell via a donor DNA construct.

The donor DNA construct further comprises a first and a second region ofhomology that flank the polynucleotide of interest. The first and secondregions of homology of the donor DNA share homology to a first and asecond genomic region, respectively, present in or flanking the targetsite of the cell or organism genome.

The donor DNA can be tethered to the guide polynucleotide. Tethereddonor DNAs can allow for co-localizing target and donor DNA, useful ingenome editing, gene insertion, and targeted genome regulation, and canalso be useful in targeting post-mitotic cells where function ofendogenous HR machinery is expected to be highly diminished (Mali etal., 2013, Nature Methods Vol. 10:957-963).

The amount of homology or sequence identity shared by a target and adonor polynucleotide can vary and includes total lengths and/or regionshaving unit integral values in the ranges of about 1-20 bp, 20-50 bp,50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp,300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb,2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including thetotal length of the target site. These ranges include every integerwithin the range, for example, the range of 1-20 bp includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. Theamount of homology can also be described by percent sequence identityover the full aligned length of the two polynucleotides which includespercent sequence identity at least of about 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,between 98% and 99%, 99%, between 99% and 100%, or 100%. Sufficienthomology includes any combination of polynucleotide length, globalpercent sequence identity, and optionally conserved regions ofcontiguous nucleotides or local percent sequence identity, for examplesufficient homology can be described as a region of 75-150 bp having atleast 80% sequence identity to a region of the target locus. Sufficienthomology can also be described by the predicted ability of twopolynucleotides to specifically hybridize under high stringencyconditions, see, for example, Sambrook et al., (1989) Molecular Cloning:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); CurrentProtocols in Molecular Biology, Ausubel et al., Eds (1994) CurrentProtocols, (Greene Publishing Associates, Inc. and John Wiley & Sons,Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, NewYork).

The structural similarity between a given genomic region and thecorresponding region of homology found on the donor DNA can be anydegree of sequence identity that allows for homologous recombination tooccur. For example, the amount of homology or sequence identity sharedby the “region of homology” of the donor DNA and the “genomic region” ofthe organism genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that thesequences undergo homologous recombination

The region of homology on the donor DNA can have homology to anysequence flanking the target site. While in some instances the regionsof homology share significant sequence homology to the genomic sequenceimmediately flanking the target site, it is recognized that the regionsof homology can be designed to have sufficient homology to regions thatmay be further 5′ or 3′ to the target site. The regions of homology canalso have homology with a fragment of the target site along withdownstream genomic regions

In one embodiment, the first region of homology further comprises afirst fragment of the target site and the second region of homologycomprises a second fragment of the target site, wherein the first andsecond fragments are dissimilar.

Polynucleotides of Interest

Polynucleotides of interest are further described herein and includepolynucleotides reflective of the commercial markets and interests ofthose involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for genetic engineering willchange accordingly.

General categories of polynucleotides of interest include, for example,genes of interest involved in information, such as zinc fingers, thoseinvolved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific polynucleotidesof interest include, but are not limited to, genes involved in traits ofagronomic interest such as but not limited to: crop yield, grainquality, crop nutrient content, starch and carbohydrate quality andquantity as well as those affecting kernel size, sucrose loading,protein quality and quantity, nitrogen fixation and/or utilization,fatty acid and oil composition, genes encoding proteins conferringresistance to abiotic stress (such as drought, nitrogen, temperature,salinity, toxic metals or trace elements, or those conferring resistanceto toxins such as pesticides and herbicides), genes encoding proteinsconferring resistance to biotic stress (such as attacks by fungi,viruses, bacteria, insects, and nematodes, and development of diseasesassociated with these organisms).

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389.

Polynucleotide sequences of interest may encode proteins involved inproviding disease or pest resistance. By “disease resistance” or “pestresistance” is intended that the plants avoid the harmful symptoms thatare the outcome of the plant-pathogen interactions. Pest resistancegenes may encode resistance to pests that have great yield drag such asrootworm, cutworm, European Corn Borer, and the like. Disease resistanceand insect resistance genes such as lysozymes or cecropins forantibacterial protection, or proteins such as defensins, glucanases orchitinases for antifungal protection, or Bacillus thuringiensisendotoxins, protease inhibitors, collagenases, lectins, or glycosidasesfor controlling nematodes or insects are all examples of useful geneproducts. Genes encoding disease resistance traits includedetoxification genes, such as against fumonisin (U.S. Pat. No.5,792,931); avirulence (avr) and disease resistance (R) genes (Jones etal. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; andMindrinos et al. (1994) Cell 78:1089); and the like. Insect resistancegenes may encode resistance to pests that have great yield drag such asrootworm, cutworm, European Corn Borer, and the like. Such genesinclude, for example, Bacillus thuringiensis toxic protein genes (U.S.Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; andGeiser et al. (1986) Gene 48:109); and the like.

An “herbicide resistance protein” or a protein resulting from expressionof an “herbicide resistance-encoding nucleic acid molecule” includesproteins that confer upon a cell the ability to tolerate a higherconcentration of an herbicide than cells that do not express theprotein, or to tolerate a certain concentration of an herbicide for alonger period of time than cells that do not express the protein.Herbicide resistance traits may be introduced into plants by genescoding for resistance to herbicides that act to inhibit the action ofacetolactate synthase (ALS, also referred to as acetohydroxyacidsynthase, AHAS), in particular the sulfonylurea (UK:sulphonylurea) typeherbicides, genes coding for resistance to herbicides that act toinhibit the action of glutamine synthase, such as phosphinothricin orbasta (e.g., the bar gene), glyphosate (e.g., the EPSP synthase gene andthe GAT gene), HPPD inhibitors (e.g, the HPPD gene) or other such genesknown in the art. See, for example, U.S. Pat. Nos. 7,626,077, 5,310,667,5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762.The bar gene encodes resistance to the herbicide basta, the nptII geneencodes resistance to the antibiotics kanamycin and geneticin, and theALS-gene mutants encode resistance to the herbicide chlorsulfuron.

Furthermore, it is recognized that the polynucleotide of interest mayalso comprise antisense sequences complementary to at least a portion ofthe messenger RNA (mRNA) for a targeted gene sequence of interest.Antisense nucleotides are constructed to hybridize with thecorresponding mRNA. Modifications of the antisense sequences may be madeas long as the sequences hybridize to and interfere with expression ofthe corresponding mRNA. In this manner, antisense constructions having70%, 80%, or 85% sequence identity to the corresponding antisensesequences may be used. Furthermore, portions of the antisensenucleotides may be used to disrupt the expression of the target gene.Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200nucleotides, or greater may be used.

In addition, the polynucleotide of interest may also be used in thesense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingpolynucleotides in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, generally greater than about 65% sequence identity,about 85% sequence identity, or greater than about 95% sequenceidentity. See U.S. Pat. Nos. 5,283,184 and 5,034,323.

The polynucleotide of interest can also be a phenotypic marker. Aphenotypic marker is screenable or a selectable marker that includesvisual markers and selectable markers whether it is a positive ornegative selectable marker. Any phenotypic marker can be used.Specifically, a selectable or screenable marker comprises a DNA segmentthat allows one to identify, or select for or against a molecule or acell that comprises it, often under particular conditions. These markerscan encode an activity, such as, but not limited to, production of RNA,peptide, or protein, or can provide a binding site for RNA, peptides,proteins, inorganic and organic compounds or compositions and the like.

Examples of selectable markers include, but are not limited to, DNAsegments that comprise restriction enzyme sites; DNA segments thatencode products which provide resistance against otherwise toxiccompounds including antibiotics, such as, spectinomycin, ampicillin,kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) andhygromycin phosphotransferase (HPT)); DNA segments that encode productswhich are otherwise lacking in the recipient cell (e.g., tRNA genes,auxotrophic markers); DNA segments that encode products which can bereadily identified (e.g., phenotypic markers such as β-galactosidase,GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan(CFP), yellow (YFP), red (RFP), and cell surface proteins); thegeneration of new primer sites for PCR (e.g., the juxtaposition of twoDNA sequence not previously juxtaposed), the inclusion of DNA sequencesnot acted upon or acted upon by a restriction endonuclease or other DNAmodifying enzyme, chemical, etc.; and, the inclusion of a DNA sequencesrequired for a specific modification (e.g., methylation) that allows itsidentification.

Additional selectable markers include genes that confer resistance toherbicidal compounds, such as sulphonylureas, glufosinate ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Seefor example, Acetolactase synthase (ALS) for resistance tosulfonylureas, imidazolinones, triazolopyrimidine sulfonamides,pyrimidinylsalicylates and sulphonylaminocarbonyl-triazolinones (Shanerand Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110);glyphosate resistant 5-enolpyruvylshikimate-3-phosphate (EPSPS) (Sarohaet al. 1998, J. Plant Biochemistry & Biotechnology Vol 7:65-72);

Polynucleotides of interest includes genes that can be stacked or usedin combination with other traits, such as but not limited to herbicideresistance or any other trait described herein. Polynucleotides ofinterest and/or traits can be stacked together in a complex trait locusas described in US20130263324 published 3 Oct. 2013 and inWO/2013/112686, published 1 Aug. 2013.

A polypeptide of interest includes any protein or polypeptide that isencoded by a polynucleotide of interest described herein.

Further provided are methods for identifying at least one plant cell,comprising in its genome, a polynucleotide of interest integrated at thetarget site. A variety of methods are available for identifying thoseplant cells with insertion into the genome at or near to the targetsite. Such methods can be viewed as directly analyzing a target sequenceto detect any change in the target sequence, including but not limitedto PCR methods, sequencing methods, nuclease digestion, Southern blots,and any combination thereof. See, for example, US20090133152 published21 May 2009. The method also comprises recovering a plant from the plantcell comprising a polynucleotide of interest integrated into its genome.The plant may be sterile or fertile. It is recognized that anypolynucleotide of interest can be provided, integrated into the plantgenome at the target site, and expressed in a plant.

Optimization of Sequences for Expression in Plants

Methods are available in the art for synthesizing plant-preferred genes.See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray etal. (1989) Nucleic Acids Res. 17:477-498. Additional sequencemodifications are known to enhance gene expression in a plant host.These include, for example, elimination of: one or more sequencesencoding spurious polyadenylation signals, one or more exon-intronsplice site signals, one or more transposon-like repeats, and other suchwell-characterized sequences that may be deleterious to gene expression.The G-C content of the sequence may be adjusted to levels average for agiven plant host, as calculated by reference to known genes expressed inthe host plant cell. When possible, the sequence is modified to avoidone or more predicted hairpin secondary mRNA structures. Thus, “aplant-optimized nucleotide sequence” of the present disclosure comprisesone or more of such sequence modifications.

Expression Elements

Any polynucleotide encoding a Cas protein or other CRISPR systemcomponent disclosed herein may be functionally linked to a heterologousexpression element, to facilitate transcription or regulation in a hostcell. Such expression elements include but are not limited to: promoter,leader, intron, and terminator. Expression elements may be“minimal”—meaning a shorter sequence derived from a native source, thatstill functions as an expression regulator or modifier. Alternatively,an expression element may be “optimized”—meaning that its polynucleotidesequence has been altered from its native state in order to functionwith a more desirable characteristic in a particular host cell (forexample, but not limited to, a bacterial promoter may be“maize-optimized” to improve its expression in corn plants).Alternatively, an expression element may be “synthetic”—meaning that itis designed in silico and synthesized for use in a host cell. Syntheticexpression elements may be entirely synthetic, or partially synthetic(comprising a fragment of a naturally-occurring polynucleotidesequence).

It has been shown that certain promoters are able to direct RNAsynthesis at a higher rate than others. These are called “strongpromoters”. Certain other promoters have been shown to direct RNAsynthesis at higher levels only in particular types of cells or tissuesand are often referred to as “tissue specific promoters”, or“tissue-preferred promoters” if the promoters direct RNA synthesispreferably in certain tissues but also in other tissues at reducedlevels.

A plant promoter includes a promoter capable of initiating transcriptionin a plant cell. For a review of plant promoters, see, Potenza et al.,2004, In vitro Cell Dev Biol 40:1-22; Porto et al., 2014, MolecularBiotechnology (2014), 56(1), 38-49.

Constitutive promoters include, for example, the core CaMV 35S promoter(Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al.,(1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) PlantMol Biol 12:619-32; ALS promoter (U.S. Pat. No. 5,659,026) and the like.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular plant tissue. Tissue-preferred promoters include,for example, WO2013103367 published 11 Jul. 2013, Kawamata et al.,(1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol GenGenet 254:337-43; Russell et al., (1997) Transgenic Res 6:157-68;Rinehart et al., (1996) Plant Physiol 112:1331-41; Van Camp et al.,(1996) Plant Physiol 112:525-35; Canevascini et al., (1996) PlantPhysiol 112:513-524; Lam, (1994) Results Probl Cell Differ 20:181-96;and Guevara-Garcia et al., (1993) Plant J 4:495-505. Leaf-preferredpromoters include, for example, Yamamoto et al., (1997) Plant J12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto etal., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka etal., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al.,(1958) EMBO J 4:2723-9; Timko et al., (1988) Nature 318:57-8.Root-preferred promoters include, for example, Hire et al., (1992) PlantMol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miaoet al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS));Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.,(1990) Plant Mol Biol 14:433-43 (root-specific promoter of A.tumefaciens mannopine synthase (MAS)); Bogusz et al., (1990) Plant Cell2:633-41 (root-specific promoters isolated from Parasponia andersoniiand Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A.rhizogenes rolC and rolD root-inducing genes); Teeri et al., (1989) EMBOJ 8:343-50 (Agrobacterium wound-induced TR1′ and TR2′ genes);VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol29:759-72); and rolB promoter (Capana et al., (1994) Plant Mol Biol25:681-91; phaseolin gene (Murai et al., (1983) Science 23:476-82;Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4).See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252;5,401,836; 5,110,732 and 5,023,179.

Seed-preferred promoters include both seed-specific promoters activeduring seed development, as well as seed-germinating promoters activeduring seed germination. See, Thompson et al., (1989) BioEssays 10:108.Seed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase); and for example those disclosed inWO2000011177 published 2 Mar. 2000 and U.S. Pat. No. 6,225,529. Fordicots, seed-preferred promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-preferred promoters include, but are notlimited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy,shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also,WO2000012733 published 9 Mar. 2000, where seed-preferred promoters fromEND1 and END2 genes are disclosed.

Chemical inducible (regulated) promoters can be used to modulate theexpression of a gene in a prokaryotic and eukaryotic cell or organismthrough the application of an exogenous chemical regulator. The promotermay be a chemical-inducible promoter, where application of the chemicalinduces gene expression, or a chemical-repressible promoter, whereapplication of the chemical represses gene expression.Chemical-inducible promoters include, but are not limited to, the maizeIn2-2 promoter, activated by benzene sulfonamide herbicide safeners (DeVeylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GSTpromoter (GST-II-27, WO1993001294 published 21 Jan. 1993), activated byhydrophobic electrophilic compounds used as pre-emergent herbicides, andthe tobacco PR-la promoter (Ono et al., (2004) Biosci Biotechnol Biochem68:803-7) activated by salicylic acid. Other chemical-regulatedpromoters include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter (Schena et al., (1991) Proc. Natl.Acad. Sci. USA 88:10421-5; McNellis et al., (1998) Plant J 14:247-257);tetracycline-inducible and tetracycline-repressible promoters (Gatz etal., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and5,789,156).

Pathogen inducible promoters induced following infection by a pathogeninclude, but are not limited to those regulating expression of PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.

A stress-inducible promoter includes the RD29A promoter (Kasuga et al.(1999) Nature Biotechnol. 17:287-91). One of ordinary skill in the artis familiar with protocols for simulating stress conditions such asdrought, osmotic stress, salt stress and temperature stress and forevaluating stress tolerance of plants that have been subjected tosimulated or naturally-occurring stress conditions.

Another example of an inducible promoter useful in plant cells, is theZmCAS1 promoter, described in US20130312137 published 21 Nov. 2013.

New promoters of various types useful in plant cells are constantlybeing discovered; numerous examples may be found in the compilation byOkamuro and Goldberg, (1989) In The Biochemistry of Plants, Vol. 115,Stumpf and Conn, eds (New York, N.Y.:Academic Press), pp. 1-82.

Modification of Genomes with Novel CRISPR-Cas System Components

As described herein, a guided Cas endonuclease can recognize, bind to aDNA target sequence and introduce a single strand (nick) ordouble-strand break. Once a single or double-strand break is induced inthe DNA, the cell's DNA repair mechanism is activated to repair thebreak. Error-prone DNA repair mechanisms can produce mutations atdouble-strand break sites. The most common repair mechanism to bring thebroken ends together is the nonhomologous end-joining (NHEJ) pathway(Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity ofchromosomes is typically preserved by the repair, but deletions,insertions, or other rearrangements (such as chromosomal translocations)are possible (Siebert and Puchta, 2002, Plant Cell 14:1121-31; Pacher etal., 2007, Genetics 175:21-9).

DNA double-strand breaks appear to be an effective factor to stimulatehomologous recombination pathways (Puchta et al., (1995) Plant Mol Biol28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta,(2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- tonine-fold increase of homologous recombination was observed betweenartificially constructed homologous DNA repeats in plants (Puchta etal., (1995) Plant Mol Biol 28:281-92). In maize protoplasts, experimentswith linear DNA molecules demonstrated enhanced homologous recombinationbetween plasmids (Lyznik et al., (1991) Mol Gen Genet 230:209-18).

Homology-directed repair (HDR) is a mechanism in cells to repairdouble-stranded and single stranded DNA breaks. Homology-directed repairincludes homologous recombination (HR) and single-strand annealing (SSA)(Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form ofHDR is called homologous recombination (HR), which has the longestsequence homology requirements between the donor and acceptor DNA. Otherforms of HDR include single-stranded annealing (SSA) andbreakage-induced replication, and these require shorter sequencehomology relative to HR. Homology-directed repair at nicks(single-stranded breaks) can occur via a mechanism distinct from HDR atdouble-strand breaks (Davis and Maizels. PNAS (0027-8424), 111 (10), p.E924-E932).

Alteration of the genome of a prokaryotic and eukaryotic cell ororganism cell, for example, through homologous recombination (HR), is apowerful tool for genetic engineering. Homologous recombination has beendemonstrated in plants (Halfter et al., (1992) Mol Gen Genet 231:186-93)and insects (Dray and Gloor, 1997, Genetics 147:689-99). Homologousrecombination has also been accomplished in other organisms. Forexample, at least 150-200 bp of homology was required for homologousrecombination in the parasitic protozoan Leishmania (Papadopoulou andDumas, (1997) Nucleic Acids Res 25:4278-86). In the filamentous fungusAspergillus nidulans, gene replacement has been accomplished with aslittle as 50 bp flanking homology (Chaveroche et al., (2000) NucleicAcids Res 28:e97). Targeted gene replacement has also been demonstratedin the ciliate Tetrahymena thermophila (Gaertig et al., (1994) NucleicAcids Res 22:5391-8). In mammals, homologous recombination has been mostsuccessful in the mouse using pluripotent embryonic stem cell lines (ES)that can be grown in culture, transformed, selected and introduced intoa mouse embryo (Watson et al., 1992, Recombinant DNA, 2nd Ed.,Scientific American Books distributed by WH Freeman & Co.).

Gene Targeting

The guide polynucleotide/Cas systems described herein can be used forgene targeting.

In general, DNA targeting can be performed by cleaving one or bothstrands at a specific polynucleotide sequence in a cell with a Casprotein associated with a suitable polynucleotide component. Once asingle or double-strand break is induced in the DNA, the cell's DNArepair mechanism is activated to repair the break via nonhomologousend-joining (NHEJ) or Homology-Directed Repair (HDR) processes which canlead to modifications at the target site.

The length of the DNA sequence at the target site can vary, andincludes, for example, target sites that are at least 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than30 nucleotides in length. It is further possible that the target sitecan be palindromic, that is, the sequence on one strand reads the samein the opposite direction on the complementary strand. The nick/cleavagesite can be within the target sequence or the nick/cleavage site couldbe outside of the target sequence. In another variation, the cleavagecould occur at nucleotide positions immediately opposite each other toproduce a blunt end cut or, in other cases, the incisions could bestaggered to produce single-stranded overhangs, also called “stickyends”, which can be either 5′ overhangs, or 3′ overhangs. Activevariants of genomic target sites can also be used. Such active variantscan comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to the given targetsite, wherein the active variants retain biological activity and henceare capable of being recognized and cleaved by an Cas endonuclease.

Assays to measure the single or double-strand break of a target site byan endonuclease are known in the art and generally measure the overallactivity and specificity of the agent on DNA substrates comprisingrecognition sites.

A targeting method herein can be performed in such a way that two ormore DNA target sites are targeted in the method, for example. Such amethod can optionally be characterized as a multiplex method. Two,three, four, five, six, seven, eight, nine, ten, or more target sitescan be targeted at the same time in certain embodiments. A multiplexmethod is typically performed by a targeting method herein in whichmultiple different RNA components are provided, each designed to guide aguide polynucleotide/Cas endonuclease complex to a unique DNA targetsite.

Gene Editing

The process for editing a genomic sequence combining DSB andmodification templates generally comprises: introducing into a host cella DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent,that recognizes a target sequence in the chromosomal sequence and isable to induce a DSB in the genomic sequence, and at least onepolynucleotide modification template comprising at least one nucleotidealteration when compared to the nucleotide sequence to be edited. Thepolynucleotide modification template can further comprise nucleotidesequences flanking the at least one nucleotide alteration, in which theflanking sequences are substantially homologous to the chromosomalregion flanking the DSB. Genome editing using DSB-inducing agents, suchas Cas-gRNA complexes, has been described, for example in US20150082478published on 19 Mar. 2015, WO2015026886 published on 26 Feb. 2015,WO2016007347 published 14 Jan. 2016, and WO/2016/025131 published on 18Feb. 2016.

Some uses for guide RNA/Cas endonuclease systems have been described(see for example: US20150082478 A1 published 19 Mar. 2015, WO2015026886published 26 Feb. 2015, and US20150059010 published 26 Feb. 2015) andinclude but are not limited to modifying or replacing nucleotidesequences of interest (such as a regulatory elements), insertion ofpolynucleotides of interest, gene knock-out, gene-knock in, modificationof splicing sites and/or introducing alternate splicing sites,modifications of nucleotide sequences encoding a protein of interest,amino acid and/or protein fusions, and gene silencing by expressing aninverted repeat into a gene of interest.

Proteins may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known. For example, amino acid sequencevariants of the protein(s) can be prepared by mutations in the DNA.Methods for mutagenesis and nucleotide sequence alterations include, forexample, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel etal., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceregarding amino acid substitutions not likely to affect biologicalactivity of the protein is found, for example, in the model of Dayhoffet al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed ResFound, Washington, D.C.). Conservative substitutions, such as exchangingone amino acid with another having similar properties, may bepreferable. Conservative deletions, insertions, and amino acidsubstitutions are not expected to produce radical changes in thecharacteristics of the protein, and the effect of any substitution,deletion, insertion, or combination thereof can be evaluated by routinescreening assays. Assays for double-strand-break-inducing activity areknown and generally measure the overall activity and specificity of theagent on DNA substrates comprising target sites.

Described herein are methods for genome editing with a Cas endonucleaseand complexes with a Cas endonuclease and a guide polynucleotide.Following characterization of the guide RNA and PAM sequence, componentsof the endonuclease and associated CRISPR RNA (crRNA) may be utilized tomodify chromosomal DNA in other organisms including plants. Tofacilitate optimal expression and nuclear localization (for eukaryoticcells), the genes comprising the complex may be optimized as describedin WO2016186953 published 24 Nov. 2016, and then delivered into cells asDNA expression cassettes by methods known in the art. The componentsnecessary to comprise an active complex may also be delivered as RNAwith or without modifications that protect the RNA from degradation oras mRNA capped or uncapped (Zhang, Y. et al., 2016, Nat. Commun.7:12617) or Cas protein guide polynucleotide complexes (WO2017070032published 27 Apr. 2017), or any combination thereof. Additionally, apart or part(s) of the complex and crRNA may be expressed from a DNAconstruct while other components are delivered as RNA with or withoutmodifications that protect the RNA from degradation or as mRNA capped oruncapped (Zhang et al. 2016 Nat. Commun. 7:12617) or Cas protein guidepolynucleotide complexes (WO2017070032 published 27 Apr. 2017) or anycombination thereof. To produce crRNAs in-vivo, tRNA derived elementsmay also be used to recruit endogenous RNAses to cleave crRNAtranscripts into mature forms capable of guiding the complex to its DNAtarget site, as described, for example, in WO2017105991 published 22Jun. 2017. Nickase complexes may be utilized separately or concertedlyto generate a single or multiple DNA nicks on one or both DNA strands.Furthermore, the cleavage activity of the Cas endonuclease may bedeactivated by altering key catalytic residues in its cleavage domain(Sinkunas, T. et al., 2013, EMBO J. 32:385-394) resulting in a RNAguided helicase that may be used to enhance homology directed repair,induce transcriptional activation, or remodel local DNA structures.Moreover, the activity of the Cas cleavage and helicase domains may bothbe knocked-out and used in combination with other DNA cutting, DNAnicking, DNA binding, transcriptional activation, transcriptionalrepression, DNA remodeling, DNA deamination, DNA unwinding, DNArecombination enhancing, DNA integration, DNA inversion, and DNA repairagents.

The transcriptional direction of the tracrRNA for the CRISPR-Cas system(if present) and other components of the CRISPR-Cas system (such asvariable targeting domain, crRNA repeat, loop, anti-repeat) can bededuced as described in WO2016186946 published 24 Nov. 2016, andWO2016186953 published 24 Nov. 2016.

As described herein, once the appropriate guide RNA requirement isestablished, the PAM preferences for each new system disclosed hereinmay be examined. If the cleavage complex results in degradation of therandomized PAM library, the complex can be converted into a nickase bydisabling the ATPase dependent helicase activity either throughmutagenesis of critical residues or by assembling the reaction in theabsence of ATP as described previously (Sinkunas, T. et al., 2013, EMBOJ. 32:385-394). Two regions of PAM randomization separated by twoprotospacer targets may be utilized to generate a double-stranded DNAbreak which may be captured and sequenced to examine the PAM sequencesthat support cleavage by the respective complex.

In one embodiment, the invention describes a method for modifying atarget site in the genome of a cell, the method comprising introducinginto a cell at least one PGEN described herein, and identifying at leastone cell that has a modification at said target, wherein themodification at said target site is selected from the group consistingof (i) a replacement of at least one nucleotide, (ii) a deletion of atleast one nucleotide, (iii) an insertion of at least one nucleotide, thechemical alteration of at least one nucleotide, and (v) any combinationof (i)-(iv).

The nucleotide to be edited can be located within or outside a targetsite recognized and cleaved by a Cas endonuclease. In one embodiment,the at least one nucleotide modification is not a modification at atarget site recognized and cleaved by a Cas endonuclease. In anotherembodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between theat least one nucleotide to be edited and the genomic target site.

A knock-out may be produced by an indel (insertion or deletion ofnucleotide bases in a target DNA sequence through NHEJ), or by specificremoval of sequence that reduces or completely destroys the function ofsequence at or near the targeting site.

A guide polynucleotide/Cas endonuclease induced targeted mutation canoccur in a nucleotide sequence that is located within or outside agenomic target site that is recognized and cleaved by the Casendonuclease.

The method for editing a nucleotide sequence in the genome of a cell canbe a method without the use of an exogenous selectable marker byrestoring function to a non-functional gene product.

In one embodiment, the invention describes a method for modifying atarget site in the genome of a cell, the method comprising introducinginto a cell at least one PGEN described herein and at least one donorDNA, wherein said donor DNA comprises a polynucleotide of interest, andoptionally, further comprising identifying at least one cell that saidpolynucleotide of interest integrated in or near said target site.

In one aspect, the methods disclosed herein may employ homologousrecombination (HR) to provide integration of the polynucleotide ofinterest at the target site.

Various methods and compositions can be employed to produce a cell ororganism having a polynucleotide of interest inserted in a target sitevia activity of a CRISPR-Cas system component described herein. In onemethod described herein, a polynucleotide of interest is introduced intothe organism cell via a donor DNA construct. As used herein, “donor DNA”is a DNA construct that comprises a polynucleotide of interest to beinserted into the target site of a Cas endonuclease. The donor DNAconstruct further comprises a first and a second region of homology thatflank the polynucleotide of interest. The first and second regions ofhomology of the donor DNA share homology to a first and a second genomicregion, respectively, present in or flanking the target site of the cellor organism genome.

The donor DNA can be tethered to the guide polynucleotide. Tethereddonor DNAs can allow for co-localizing target and donor DNA, useful ingenome editing, gene insertion, and targeted genome regulation, and canalso be useful in targeting post-mitotic cells where function ofendogenous HR machinery is expected to be highly diminished (Mali etal., 2013, Nature Methods Vol. 10:957-963).

The amount of homology or sequence identity shared by a target and adonor polynucleotide can vary and includes total lengths and/or regionshaving unit integral values in the ranges of about 1-20 bp, 20-50 bp,50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp,300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb,2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including thetotal length of the target site. These ranges include every integerwithin the range, for example, the range of 1-20 bp includes 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. Theamount of homology can also be described by percent sequence identityover the full aligned length of the two polynucleotides which includespercent sequence identity of about at least 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100%. Sufficient homology includes any combination ofpolynucleotide length, global percent sequence identity, and optionallyconserved regions of contiguous nucleotides or local percent sequenceidentity, for example sufficient homology can be described as a regionof 75-150 bp having at least 80% sequence identity to a region of thetarget locus. Sufficient homology can also be described by the predictedability of two polynucleotides to specifically hybridize under highstringency conditions, see, for example, Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds(1994) Current Protocols, (Greene Publishing Associates, Inc. and JohnWiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, (Elsevier, New York).

Episomal DNA molecules can also be ligated into the double-strand break,for example, integration of T-DNAs into chromosomal double-strand breaks(Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta,(1998) EMBO J. 17:6086-95). Once the sequence around the double-strandbreaks is altered, for example, by exonuclease activities involved inthe maturation of double-strand breaks, gene conversion pathways canrestore the original structure if a homologous sequence is available,such as a homologous chromosome in non-dividing somatic cells, or asister chromatid after DNA replication (Molinier et al., (2004) PlantCell 16:342-52). Ectopic and/or epigenic DNA sequences may also serve asa DNA repair template for homologous recombination (Puchta, (1999)Genetics 152:1173-81).

In one embodiment, the disclosure comprises a method for editing anucleotide sequence in the genome of a cell, the method comprisingintroducing into at least one PGEN described herein, and apolynucleotide modification template, wherein said polynucleotidemodification template comprises at least one nucleotide modification ofsaid nucleotide sequence, and optionally further comprising selecting atleast one cell that comprises the edited nucleotide sequence.

The guide polynucleotide/Cas endonuclease system can be used incombination with at least one polynucleotide modification template toallow for editing (modification) of a genomic nucleotide sequence ofinterest. (See also US20150082478, published 19 Mar. 2015 andWO2015026886 published 26 Feb. 2015).

Polynucleotides of interest and/or traits can be stacked together in acomplex trait locus as described in WO2012129373 published 27 Sep. 2012,and in WO2013112686, published 1 Aug. 2013. The guidepolynucleotide/Cas9 endonuclease system described herein provides for anefficient system to generate double-strand breaks and allows for traitsto be stacked in a complex trait locus.

A guide polynucleotide/Cas system as described herein, mediating genetargeting, can be used in methods for directing heterologous geneinsertion and/or for producing complex trait loci comprising multipleheterologous genes in a fashion similar as disclosed in WO2012129373published 27 Sep. 2012, where instead of using a double-strand breakinducing agent to introduce a gene of interest, a guidepolynucleotide/Cas system as disclosed herein is used. By insertingindependent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5centimorgans (cM) from each other, the transgenes can be bred as asingle genetic locus (see, for example, US20130263324 published 3 Oct.2013 or WO2012129373 published 14 Mar. 2013). After selecting a plantcomprising a transgene, plants comprising (at least) one transgenes canbe crossed to form an F1 that comprises both transgenes. In progeny fromthese F1 (F2 or BC1) 1/500 progeny would have the two differenttransgenes recombined onto the same chromosome. The complex locus canthen be bred as single genetic locus with both transgene traits. Thisprocess can be repeated to stack as many traits as desired.

Further uses for guide RNA/Cas endonuclease systems have been described(See for example: US20150082478 published 19 Mar. 2015, WO2015026886published 26 Feb. 2015, US20150059010 published 26 Feb. 2015,WO2016007347 published 14 Jan. 2016, and PCT application WO2016025131published 18 Feb. 2016) and include but are not limited to modifying orreplacing nucleotide sequences of interest (such as a regulatoryelements), insertion of polynucleotides of interest, gene knock-out,gene-knock in, modification of splicing sites and/or introducingalternate splicing sites, modifications of nucleotide sequences encodinga protein of interest, amino acid and/or protein fusions, and genesilencing by expressing an inverted repeat into a gene of interest.

Resulting characteristics from the gene editing compositions and methodsdescribed herein may be evaluated. Chromosomal intervals that correlatewith a phenotype or trait of interest can be identified. A variety ofmethods well known in the art are available for identifying chromosomalintervals. The boundaries of such chromosomal intervals are drawn toencompass markers that will be linked to the gene controlling the traitof interest. In other words, the chromosomal interval is drawn such thatany marker that lies within that interval (including the terminalmarkers that define the boundaries of the interval) can be used as amarker for a particular trait. In one embodiment, the chromosomalinterval comprises at least one QTL, and furthermore, may indeedcomprise more than one QTL. Close proximity of multiple QTLs in the sameinterval may obfuscate the correlation of a particular marker with aparticular QTL, as one marker may demonstrate linkage to more than oneQTL. Conversely, e.g., if two markers in close proximity showco-segregation with the desired phenotypic trait, it is sometimesunclear if each of those markers identifies the same QTL or twodifferent QTL. The term “quantitative trait locus” or “QTL” refers to aregion of DNA that is associated with the differential expression of aquantitative phenotypic trait in at least one genetic background, e.g.,in at least one breeding population. The region of the QTL encompassesor is closely linked to the gene or genes that affect the trait inquestion. An “allele of a QTL” can comprise multiple genes or othergenetic factors within a contiguous genomic region or linkage group,such as a haplotype. An allele of a QTL can denote a haplotype within aspecified window wherein said window is a contiguous genomic region thatcan be defined, and tracked, with a set of one or more polymorphicmarkers. A haplotype can be defined by the unique fingerprint of allelesat each marker within the specified window.

Introduction of CRISPR-Cas System Components into a Cell

The methods and compositions described herein do not depend on aparticular method for introducing a sequence into an organism or cell,only that the polynucleotide or polypeptide gains access to the interiorof at least one cell of the organism. Introducing includes reference tothe incorporation of a nucleic acid into a eukaryotic or prokaryoticcell where the nucleic acid may be incorporated into the genome of thecell, and includes reference to the transient (direct) provision of anucleic acid, protein or polynucleotide-protein complex (PGEN, RGEN) tothe cell.

Methods for introducing polynucleotides or polypeptides or apolynucleotide-protein complex into cells or organisms are known in theart including, but not limited to, microinjection, electroporation,stable transformation methods, transient transformation methods,ballistic particle acceleration (particle bombardment), whiskersmediated transformation, Agrobacterium-mediated transformation, directgene transfer, viral-mediated introduction, transfection, transduction,cell-penetrating peptides, mesoporous silica nanoparticle (MSN)-mediateddirect protein delivery, topical applications, sexual crossing, sexualbreeding, and any combination thereof.

For example, the guide polynucleotide (guide RNA,crNucleotide+tracrNucleotide, guide DNA and/or guide RNA-DNA molecule)can be introduced into a cell directly (transiently) as a singlestranded or double stranded polynucleotide molecule. The guide RNA (orcrRNA+tracrRNA) can also be introduced into a cell indirectly byintroducing a recombinant DNA molecule comprising a heterologous nucleicacid fragment encoding the guide RNA (or crRNA+tracrRNA), operablylinked to a specific promoter that is capable of transcribing the guideRNA (crRNA+tracrRNA molecules) in said cell. The specific promoter canbe, but is not limited to, a RNA polymerase III promoter, which allowfor transcription of RNA with precisely defined, unmodified, 5′- and3′-ends (Ma et al., 2014, Mol. Ther. Nucleic Acids 3:e161; DiCarlo etal., 2013, Nucleic Acids Res. 41:4336-4343; WO2015026887, published 26Feb. 2015). Any promoter capable of transcribing the guide RNA in a cellcan be used and includes a heat shock/heat inducible promoter operablylinked to a nucleotide sequence encoding the guide RNA.

Plant cells differ from animal cells (such as human cells), fungal cells(such as yeast cells) and protoplasts, including for example plant cellscomprise a plant cell wall which may act as a barrier to the delivery ofcomponents.

Delivery of the Cas endonuclease, and/or the guide RNA, and/or aribonucleoprotein complex, and/or a polynucleotide encoding any one ormore of the preceding, into plant cells can be achieved through methodsknown in the art, for example but not limited to: Rhizobiales-mediatedtransformation (e.g., Agrobacterium, Ochrobactrum), particle mediateddelivery (particle bombardment), polyethylene glycol (PEG)-mediatedtransfection (for example to protoplasts), electroporation,cell-penetrating peptides, or mesoporous silica nanoparticle(MSN)-mediated direct protein delivery.

The Cas endonuclease, such as the Cas endonuclease described herein, canbe introduced into a cell by directly introducing the Cas polypeptideitself (referred to as direct delivery of Cas endonuclease), the mRNAencoding the Cas protein, and/or the guide polynucleotide/Casendonuclease complex itself, using any method known in the art. The Casendonuclease can also be introduced into a cell indirectly byintroducing a recombinant DNA molecule that encodes the Casendonuclease. The endonuclease can be introduced into a cell transientlyor can be incorporated into the genome of the host cell using any methodknown in the art. Uptake of the endonuclease and/or the guidedpolynucleotide into the cell can be facilitated with a Cell PenetratingPeptide (CPP) as described in WO2016073433 published 12 May 2016. Anypromoter capable of expressing the Cas endonuclease in a cell can beused and includes a heat shock/heat inducible promoter operably linkedto a nucleotide sequence encoding the Cas endonuclease.

Direct delivery of a polynucleotide modification template into plantcells can be achieved through particle mediated delivery, and any otherdirect method of delivery, such as but not limiting to, polyethyleneglycol (PEG)-mediated transfection to protoplasts, whiskers mediatedtransformation, electroporation, particle bombardment, cell-penetratingpeptides, or mesoporous silica nanoparticle (MSN)-mediated directprotein delivery can be successfully used for delivering apolynucleotide modification template in eukaryotic cells, such as plantcells.

The donor DNA can be introduced by any means known in the art. The donorDNA may be provided by any transformation method known in the artincluding, for example, Agrobacterium-mediated transformation orbiolistic particle bombardment. The donor DNA may be present transientlyin the cell or it could be introduced via a viral replicon. In thepresence of the Cas endonuclease and the target site, the donor DNA isinserted into the transformed plant's genome.

Direct delivery of any one of the guided Cas system components can beaccompanied by direct delivery (co-delivery) of other mRNAs that canpromote the enrichment and/or visualization of cells receiving the guidepolynucleotide/Cas endonuclease complex components. For example, directco-delivery of the guide polynucleotide/Cas endonuclease components(and/or guide polynucleotide/Cas endonuclease complex itself) togetherwith mRNA encoding phenotypic markers (such as but not limiting totranscriptional activators such as CRC (Bruce et al. 2000 The Plant Cell12:65-79) can enable the selection and enrichment of cells without theuse of an exogenous selectable marker by restoring function to anon-functional gene product as described in WO2017070032 published 27Apr. 2017.

Introducing a guide RNA/Cas endonuclease complex described herein,(representing the cleavage ready complex described herein) into a cellincludes introducing the individual components of said complex eitherseparately or combined into the cell, and either directly (directdelivery as RNA for the guide and protein for the Cas endonuclease andprotein subunits, or functional fragments thereof) or via recombinationconstructs expressing the components (guide RNA, Cas endonuclease,protein subunits, or functional fragments thereof). Introducing a guideRNA/Cas endonuclease complex (RGEN) into a cell includes introducing theguide RNA/Cas endonuclease complex as a ribonucleotide-protein into thecell. The ribonucleotide-protein can be assembled prior to beingintroduced into the cell as described herein. The components comprisingthe guide RNA/Cas endonuclease ribonucleotide protein (at least one Casendonuclease, at least one guide RNA, at least one protein subunit) canbe assembled in vitro or assembled by any means known in the art priorto being introduced into a cell (targeted for genome modification asdescribed herein).

Direct delivery of the RGEN ribonucleoprotein, allows for genome editingat a target site in the genome of a cell which can be followed by rapiddegradation of the complex, and only a transient presence of the complexin the cell. This transient presence of the RGEN complex may lead toreduced off-target effects. In contrast, delivery of RGEN components(guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result inconstant expression of RGENs from these plasmids which can intensify offtarget effects (Cradick, T. J. et al. (2013) Nucleic Acids Res41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. 31:822-826).

Direct delivery can be achieved by combining any one component of theguide RNA/Cas endonuclease complex (RGEN), representing the cleavageready complex described herein, (such as at least one guide RNA, atleast one Cas protein, and optionally one additional protein), with adelivery matrix comprising a microparticle (such as but not limited toof a gold particle, tungsten particle, and silicon carbide whiskerparticle) (see also WO2017070032 published 27 Apr. 2017). The deliverymatrix may comprise any one of the components, such as the Casendonuclease, that is attached to a solid matrix (e.g., a particle forbombardment).

In one aspect the guide polynucleotide/Cas endonuclease complex, is acomplex wherein the guide RNA and Cas endonuclease protein forming theguide RNA/Cas endonuclease complex are introduced into the cell as RNAand protein, respectively.

In one aspect the guide polynucleotide/Cas endonuclease complex, is acomplex wherein the guide RNA and Cas endonuclease protein and the atleast one protein subunit of a complex forming the guide RNA/Casendonuclease complex are introduced into the cell as RNA and proteins,respectively.

In one aspect the guide polynucleotide/Cas endonuclease complex, is acomplex wherein the guide RNA and Cas endonuclease protein and the atleast one protein subunit of a complex forming the guide RNA/Casendonuclease complex (cleavage ready complex) are preassembled in vitroand introduced into the cell as a ribonucleotide-protein complex.

Protocols for introducing polynucleotides, polypeptides orpolynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells,such as plants or plant cells are known and include microinjection(Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No.6,300,543), meristem transformation (U.S. Pat. No. 5,736,369),electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA83:5602-6, Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055 and 5,981,840), whiskers mediated transformation (Ainley etal. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M.Arshad 2011 Properties and Applications of Silicon Carbide (2011),345-358 Editor(s):Gerhardt, Rosario. Publisher: InTech, Rijeka, Croatia.CODEN:69PQBP; ISBN:978-953-307-201-2), direct gene transfer (Paszkowskiet al., (1984) EMBO J 3:2717-22), and ballistic particle acceleration(U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes etal., (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg & Phillips (Springer-Verlag, Berlin);McCabe et al., (1988) Biotechnology 6:923-6; Weissinger et al., (1988)Ann Rev Genet 22:421-77; Sanford et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al., (1988) Plant Physiol87:671-4 (soybean); Finer and McMullen, (1991) In vitro Cell Dev Biol27P:175-82 (soybean); Singh et al., (1998) Theor Appl Genet 96:319-24(soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein etal., (1988) Proc. Natl. Acad. Sci. USA 85:4305-9 (maize); Klein et al.,(1988) Biotechnology 6:559-63 (maize); U.S. Pat. Nos. 5,240,855;5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 91:440-4(maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize);Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Pat. No.5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA84:5345-9 (Liliaceae); De Wet et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al., (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) andKaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediatedtransformation); D'Halluin et al., (1992) Plant Cell 4:1495-505(electroporation); Li et al., (1993) Plant Cell Rep 12:250-5; Christouand Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996)Nat Biotechnol 14:745-50 (maize via Agrobacterium tumefaciens).

Alternatively, polynucleotides may be introduced into plant or plantcells by contacting cells or organisms with a virus or viral nucleicacids. Generally, such methods involve incorporating a polynucleotidewithin a viral DNA or RNA molecule. In some examples a polypeptide ofinterest may be initially synthesized as part of a viral polyprotein,which is later processed by proteolysis in vivo or in vitro to producethe desired recombinant protein. Methods for introducing polynucleotidesinto plants and expressing a protein encoded therein, involving viralDNA or RNA molecules, are known, see, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.

The polynucleotide or recombinant DNA construct can be provided to orintroduced into a prokaryotic and eukaryotic cell or organism using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, the introductionof the polynucleotide construct directly into the plant.

Nucleic acids and proteins can be provided to a cell by any methodincluding methods using molecules to facilitate the uptake of anyone orall components of a guided Cas system (protein and/or nucleic acids),such as cell-penetrating peptides and nanocarriers. See alsoUS20110035836 published 10 Feb. 2011, and EP2821486A1 published 7 Jan.2015.

Other methods of introducing polynucleotides into a prokaryotic andeukaryotic cell or organism or plant part can be used, including plastidtransformation methods, and the methods for introducing polynucleotidesinto tissues from seedlings or mature seeds.

Stable transformation is intended to mean that the nucleotide constructintroduced into an organism integrates into a genome of the organism andis capable of being inherited by the progeny thereof. Transienttransformation is intended to mean that a polynucleotide is introducedinto the organism and does not integrate into a genome of the organismor a polypeptide is introduced into an organism. Transienttransformation indicates that the introduced composition is onlytemporarily expressed or present in the organism.

A variety of methods are available to identify those cells having analtered genome at or near a target site without using a screenablemarker phenotype. Such methods can be viewed as directly analyzing atarget sequence to detect any change in the target sequence, includingbut not limited to PCR methods, sequencing methods, nuclease digestion,Southern blots, and any combination thereof.

Cells and Plants

The presently disclosed polynucleotides and polypeptides can beintroduced into a cell. Cells include, but are not limited to, human,non-human, animal, mammalian, bacterial, fungal, insect, yeast,non-conventional yeast, and plant cells as well as plants and seedsproduced by the methods described herein. Any plant can be used with thecompositions and methods described herein, including monocot and dicotplants, and plant elements.

Examples of monocot plants that can be used include, but are not limitedto, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum(Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), wheat (Triticumspecies, for example Triticum aestivum, Triticum monococcum), sugarcane(Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicumvirgatum), pineapple (Ananas comosus), banana (Musa spp.), palm,ornamentals, turfgrasses, and other grasses.

Examples of dicot plants that can be used include, but are not limitedto, soybean (Glycine max), Brassica species (for example but not limitedto: oilseed rape or Canola) (Brassica napus, B. campestris, Brassicarapa, Brassica. juncea), alfalfa (Medicago sativa),), tobacco (Nicotianatabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthusannuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut(Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanumtuberosum.

Additional plants that can be used include safflower (Carthamustinctorius), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees(Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana(Musa spp.), avocado (Persea americana), fig (Ficus casica), guava(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),papaya (Carica papaya), cashew (Anacardium occidentale), macadamia(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Betavulgaris), vegetables, ornamentals, and conifers.

Vegetables that can be used include tomatoes (Lycopersicon esculentum),lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), limabeans (Phaseolus limensis), peas (Lathyrus spp.), and members of thegenus Cucumis such as cucumber (C. sativus), cantaloupe (C.cantalupensis), and musk melon (C. melo). Ornamentals include azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum.

Conifers that may be used include pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata);Douglas fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow cedar(Chamaecyparis nootkatensis).

In certain embodiments of the disclosure, a fertile plant is a plantthat produces viable male and female gametes and is self-fertile. Such aself-fertile plant can produce a progeny plant without the contributionfrom any other plant of a gamete and the genetic material comprisedtherein. Other embodiments of the disclosure can involve the use of aplant that is not self-fertile because the plant does not produce malegametes, or female gametes, or both, that are viable or otherwisecapable of fertilization.

The present disclosure finds use in the breeding of plants comprisingone or more introduced traits, or edited genomes.

A non-limiting example of how two traits can be stacked into the genomeat a genetic distance of, for example, 5 cM from each other is describedas follows: A first plant comprising a first transgenic target siteintegrated into a first DSB target site within the genomic window andnot having the first genomic locus of interest is crossed to a secondtransgenic plant, comprising a genomic locus of interest at a differentgenomic insertion site within the genomic window and the second plantdoes not comprise the first transgenic target site. About 5% of theplant progeny from this cross will have both the first transgenic targetsite integrated into a first DSB target site and the first genomic locusof interest integrated at different genomic insertion sites within thegenomic window. Progeny plants having both sites in the defined genomicwindow can be further crossed with a third transgenic plant comprising asecond transgenic target site integrated into a second DSB target siteand/or a second genomic locus of interest within the defined genomicwindow and lacking the first transgenic target site and the firstgenomic locus of interest. Progeny are then selected having the firsttransgenic target site, the first genomic locus of interest and thesecond genomic locus of interest integrated at different genomicinsertion sites within the genomic window. Such methods can be used toproduce a transgenic plant comprising a complex trait locus having atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic targetsites integrated into DSB target sites and/or genomic loci of interestintegrated at different sites within the genomic window. In such amanner, various complex trait loci can be generated.

Cells and Animals

The presently disclosed polynucleotides and polypeptides can beintroduced into an animal cell. Animal cells can include, but are notlimited to: an organism of a phylum including chordates, arthropods,mollusks, annelids, cnidarians, or echinoderms; or an organism of aclass including mammals, insects, birds, amphibians, reptiles, orfishes. In some aspects, the animal is human, mouse, C. elegans, rat,fruit fly (Drosophila spp.), zebrafish, chicken, dog, cat, guinea pig,hamster, chicken, Japanese ricefish, sea lamprey, pufferfish, tree frog(e.g., Xenopus spp.), monkey, or chimpanzee. Particular cell types thatare contemplated include haploid cells, diploid cells, reproductivecells, neurons, muscle cells, endocrine or exocrine cells, epithelialcells, muscle cells, tumor cells, embryonic cells, hematopoietic cells,bone cells, germ cells, somatic cells, stem cells, pluripotent stemcells, induced pluripotent stem cells, progenitor cells, meiotic cells,and mitotic cells. In some aspects, a plurality of cells from anorganism may be used.

The novel Cas9 orthologs disclosed may be used to edit the genome of ananimal cell in various ways. In one aspect, it may be desirable todelete one or more nucleotides. In another aspect, it may be desirableto insert one or more nucleotides. In one aspect, it may be desirable toreplace one or more nucleotides. In another aspect, it may be desirableto modify one or more nucleotides via a covalent or non-covalentinteraction with another atom or molecule.

Genome modification via a Cas9 ortholog may be used to effect agenotypic and/or phenotypic change on the target organism. Such a changeis preferably related to an improved phenotype of interest or aphysiologically-important characteristic, the correction of anendogenous defect, or the expression of some type of expression marker.In some aspects, the phenotype of interest or physiologically-importantcharacteristic is related to the overall health, fitness, or fertilityof the animal, the ecological fitness of the animal, or the relationshipor interaction of the animal with other organisms in its environment. Insome aspects, the phenotype of interest or physiologically-importantcharacteristic is selected from the group consisting of: improvedgeneral health, disease reversal, disease modification, diseasestabilization, disease prevention, treatment of parasitic infections,treatment of viral infections, treatment of retroviral infections,treatment of bacterial infections, treatment of neurological disorders(for example but not limited to: multiple sclerosis), correction ofendogenous genetic defects (for example but not limited to: metabolicdisorders, Achondroplasia, Alpha-1 Antitrypsin Deficiency,Antiphospholipid Syndrome, Autism, Autosomal Dominant Polycystic KidneyDisease, Barth syndrome, Breast cancer, Charcot-Marie-Tooth, Coloncancer, Cri du chat, Crohn's Disease, Cystic fibrosis, Dercum Disease,Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor VLeiden Thrombophilia, Familial Hypercholesterolemia, FamilialMediterranean Fever, Fragile X Syndrome, Gaucher Disease,Hemochromatosis, Hemophilia, Holoprosencephaly, Huntington's disease,Klinefelter syndrome, Marfan syndrome, Myotonic Dystrophy,Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson'sdisease, Phenylketonuria, Poland Anomaly, Porphyria, Progeria, ProstateCancer, Retinitis Pigmentosa, Severe Combined Immunodeficiency (SCID),Sickle cell disease, Skin Cancer, Spinal Muscular Atrophy, Tay-Sachs,Thalassemia, Trimethylaminuria, Turner Syndrome, VelocardiofacialSyndrome, WAGR Syndrome, and Wilson Disease), treatment of innate immunedisorders (for example but not limited to: immunoglobulin subclassdeficiencies), treatment of acquired immune disorders (for example butnot limited to: AIDS and other HIV-related disorders), treatment ofcancer, as well as treatment of diseases, including rare or “orphan”conditions, that have eluded effective treatment options with othermethods.

Cells that have been genetically modified using the compositions ormethods disclosed herein may be transplanted to a subject for purposessuch as gene therapy, e.g. to treat a disease, or as an antiviral,antipathogenic, or anticancer therapeutic, for the production ofgenetically modified organisms in agriculture, or for biologicalresearch.

In Vitro Polynucleotide Detection, Binding, and Modification

The compositions disclosed herein may further be used as compositionsfor use in in vitro methods, in some aspects with isolatedpolynucleotide sequence(s). Said isolated polynucleotide sequence(s) maycomprise one or more target sequence(s) for modification. In someaspects, said isolated polynucleotide sequence(s) may be genomic DNA, aPCR product, or a synthesized oligonucleotide.

Compositions

Modification of a target sequence may be in the form of a nucleotideinsertion, a nucleotide deletion, a nucleotide substitution, theaddition of an atom molecule to an existing nucleotide, a nucleotidemodification, or the binding of a heterologous polynucleotide orpolypeptide to said target sequence. The insertion of one or morenucleotides may be accomplished by the inclusion of a donorpolynucleotide in the reaction mixture: said donor polynucleotide isinserted into a double-strand break created by said Cas-alpha orthologpolypeptide. The insertion may be via non-homologous end joining or viahomologous recombination.

In one aspect, the sequence of the target polynucleotide is known priorto modification, and compared to the sequence(s) of polynucleotide(s)that result from treatment with the Cas-alpha ortholog. In one aspect,the sequence of the target polynucleotide is not known prior tomodification, and the treatment with the Cas-alpha ortholog is used aspart of a method to determine the sequence of said targetpolynucleotide.

Polynucleotide modification with a Cas-alpha ortholog may beaccomplished by usage of a full-length polypeptide identified from a Caslocus, or from a fragment, modification, or variant of a polypeptideidentified from a Cas locus. In some aspects, said Cas-alpha ortholog isobtained or derived from an organism listed in Table 1. In some aspects,said Cas-alpha ortholog is a polypeptide sharing at least 80% identitywith any of SEQ ID NOs:86-170 or 511-1135. In some aspects, saidCas-alpha ortholog is a functional variant of any of SEQ ID NOs:86-170or 511-1135. In some aspects, said Cas-alpha ortholog is a functionalfragment of any of SEQ ID NOs:86-170 or 511-1135. In some aspects, saidCas-alpha ortholog is a Cas-alpha polypeptide encoded by apolynucleotide selected from the group consisting of: SEQ ID NO:86-170or 511-1135. In some aspects, said Cas-alpha ortholog is a Cas-alphapolypeptide that recognizes a PAM sequence listed in any of Tables 4-83.In some aspects, said Cas-alpha ortholog is a Cas-alpha polypeptideidentified from an organism listed in the sequence listing.

In some aspects, the Cas-alpha ortholog is provided as a Cas-alphapolynucleotide. In some aspects, said Cas-alpha polynucleotide isselected from the group consisting of: SEQ ID NO:1-85, or a sequencesharing at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% with any one ofSEQ ID NO:1-85.

In some aspects, the Cas-alpha ortholog may be selected from the groupconsisting of: an unmodified wild type Cas-alpha ortholog, a functionalCas-alpha ortholog variant, a functional Cas-alpha ortholog fragment, afusion protein comprising an active or deactivated Cas-alpha ortholog, aCas-alpha ortholog further comprising one or more nuclear localizationsequences (NLS) on the C-terminus or on the N-terminus or on both the N-and C-termini, a biotinylated Cas-alpha ortholog, a Cas-alpha ortholognickase, a Cas-alpha ortholog endonuclease, a Cas-alpha ortholog furthercomprising a Histidine tag, and a mixture of any two or more of thepreceding.

In some aspects, the Cas-alpha ortholog is a fusion protein furthercomprising a nuclease domain, a transcriptional activator domain, atranscriptional repressor domain, an epigenetic modification domain, acleavage domain, a nuclear localization signal, a cell-penetratingdomain, a translocation domain, a marker, or a transgene that isheterologous to the target polynucleotide sequence or to the cell fromwhich said target polynucleotide sequence is obtained or derived.

In some aspects, a plurality of Cas-alpha orthologs may be desired. Insome aspects, said plurality may comprise Cas-alpha orthologs derivedfrom different source organisms or from different loci within the sameorganism. In some aspects, said plurality may comprise Cas-alphaorthologs with different binding specificities to the targetpolynucleotide. In some aspects, said plurality may comprise Cas-alphaorthologs with different cleavage efficiencies. In some aspects, saidplurality may comprise Cas-alpha orthologs with different PAMspecificities. In some aspects, said plurality may comprise orthologs ofdifferent molecular compositions, i.e., a polynucleotide Cas-alphaortholog and a polypeptide Cas-alpha ortholog.

The guide polynucleotide may be provided as a single guide RNA (sgRNA),a chimeric molecule comprising a tracrRNA, a chimeric moleculecomprising a crRNA, a chimeric RNA-DNA molecule, a DNA molecule, or apolynucleotide comprising one or more chemically modified nucleotides.

The storage conditions of the Cas-alpha ortholog and/or the guidepolynucleotide include parameters for temperature, state of matter, andtime. In some aspects, the Cas-alpha ortholog and/or the guidepolynucleotide is stored at about −80 degrees Celsius, at about −20degrees Celsius, at about 4 degrees Celsius, at about 20-25 degreesCelsius, or at about 37 degrees Celsius. In some aspects, the Cas-alphaortholog and/or the guide polynucleotide is stored as a liquid, a frozenliquid, or as a lyophilized powder. In some aspects, the Cas-alphaortholog and/or the guide polynucleotide is stable for at least one day,at least one week, at least one month, at least one year, or evengreater than one year.

Any or all of the possible polynucleotide components of the reaction(e.g., guide polynucleotide, donor polynucleotide, optionally aCas-alpha polynucleotide) may be provided as part of a vector, aconstruct, a linearized or circularized plasmid, or as part of achimeric molecule. Each component may be provided to the reactionmixture separately or together. In some aspects, one or more of thepolynucleotide components are operably linked to a heterologousnoncoding regulatory element that regulates its expression.

The method for modification of a target polynucleotide comprisescombining the minimal elements into a reaction mixture comprising: aCas-alpha ortholog (or variant, fragment, or other related molecule asdescribed above), a guide polynucleotide comprising a sequence that issubstantially complementary to, or selectively hybridizes to, the targetpolynucleotide sequence of the target polynucleotide, and a targetpolynucleotide for modification. In some aspects, the Cas-alpha orthologis provided as a polypeptide. In some aspects, the Cas-alpha ortholog isprovided as a Cas-alpha ortholog polynucleotide. In some aspects, theguide polynucleotide is provided as an RNA molecule, a DNA molecule, anRNA:DNA hybrid, or a polynucleotide molecule comprising achemically-modified nucleotide.

The storage buffer of any one of the components, or the reactionmixture, may be optimized for stability, efficacy, or other parameters.Additional components of the storage buffer or the reaction mixture mayinclude a buffer composition, Tris, EDTA, dithiothreitol (DTT),phosphate-buffered saline (PBS), sodium chloride, magnesium chloride,HEPES, glycerol, BSA, a salt, an emulsifier, a detergent, a chelatingagent, a redox reagent, an antibody, nuclease-free water, a proteinase,and/or a viscosity agent. In some aspects, the storage buffer orreaction mixture further comprises a buffer solution with at least oneof the following components: HEPES, MgCl2, NaCl, EDTA, a proteinase,Proteinase K, glycerol, nuclease-free water.

Incubation conditions will vary according to desired outcome. Thetemperature is preferably at least 10 degrees Celsius, between 10 and15, at least 15, between 15 and 17, at least 17, between 17 and 20, atleast 20, between 20 and 22, at least 22, between 22 and 25, at least25, between 25 and 27, at least 27, between 27 and 30, at least 30,between 30 and 32, at least 32, between 32 and 35, at least 35, at least36, at least 37, at least 38, at least 39, at least 40, or even greaterthan 40 degrees Celsius. The time of incubation is at least 1 minute, atleast 2 minutes, at least 3 minutes, at least 4 minutes, at least 5minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, atleast 9 minutes, at least 10 minutes, or even greater than 10 minutes.

The sequence(s) of the polynucleotide(s) in the reaction mixture priorto, during, or after incubation may be determined by any method known inthe art. In one aspect, modification of a target polynucleotide may beascertained by comparing the sequence(s) of the polynucleotide(s)purified from the reaction mixture to the sequence of the targetpolynucleotide prior to combining with the Cas-alpha ortholog.

Any one or more of the compositions disclosed herein, useful for invitro or in vivo polynucleotide detection, binding, and/or modification,may be comprised within a kit. A kit comprises a Cas-alpha ortholog or apolynucleotide Cas-alpha ortholog encoding such, optionally furthercomprising buffer components to enable efficient storage, and one ormore additional compositions that enable the introduction of saidCas-alpha ortholog or Cas-alpha ortholog to a heterologouspolynucleotide, wherein said Cas-alpha ortholog or Cas-alpha ortholog iscapable of effecting a modification, addition, deletion, or substitutionof at least one nucleotide of said heterologous polynucleotide. In anadditional aspect, a Cas-alpha ortholog disclosed herein may be used forthe enrichment of one or more polynucleotide target sequences from amixed pool. In an additional aspect, a Cas-alpha ortholog disclosedherein may be immobilized on a matrix for use in in vitro targetpolynucleotide detection, binding, and/or modification.

A Cas-alpha endonuclease may be attached, associated with, or affixed toa solid matrix for the purposes of storage, purification, and/orcharacterization. Examples of a solid matrix include, but are notlimited to: a filter, a chromatography resin, an assay plate, a testtube, a cryogenic vial, etc. A Cas-alpha endonuclease may besubstantially purified and stored in an appropriate buffer solution, orlyophilized.

Methods of Detection

Methods of detecting the Cas-alpha:guide polynucleotide complex bound tothe target polynucleotide may include any known in the art, includingbut not limited to microscopy, chromatographic separation,electrophoresis, immunoprecipitation, filtration, nanopore separation,microarrays, as well as those described below.

A DNA Electrophoretic Mobility Shift Assay (EMSA): studies proteinsbinding to known DNA oligonucleotide probes and assesses the specificityof the interaction. The technique is based on the principle thatprotein-DNA complexes migrate more slowly than free DNA molecules whensubjected to polyacrylamide or agarose gel electrophoresis. Because therate of DNA migration is retarded upon protein binding, the assay isalso called a gel retardation assay. Adding a protein-specific antibodyto the binding components creates an even larger complex(antibody-protein-DNA) which migrates even slower duringelectrophoresis, this is known as a supershift and can be used toconfirm protein identities.

DNA Pull-down Assays use a DNA probe labelled with a high affinity tag,such as biotin, which allows the probe to be recovered or immobilized. ADNA probe can be complexed with a protein from a cell lysate in areaction similar to that used in the EMSA and then used to purify thecomplex using agarose or magnetic beads. The proteins are then elutedfrom the DNA and detected by Western blot or identified by massspectrometry. Alternatively, the protein may be labelled with anaffinity tag or the DNA-protein complex may be isolated using anantibody against the protein of interest (similar to a supershiftassay). In this case, the unknown DNA sequence bound by the protein isdetected by Southern blotting or through PCR analysis.

Reporter assays provide a real-time in vivo read-out of translationalactivity for a promoter of interest. Reporter genes are fusions of atarget promoter DNA sequence and a reporter gene DNA sequence which iscustomized by the researcher and the DNA sequence codes for a proteinwith detectable properties like firefly/Renilla luciferase or alkalinephosphatase. These genes produce enzymes only when the promoter ofinterest is activated. The enzyme, in turn, catalyses a substrate toproduce either light or a colour change that can be detected byspectroscopic instrumentation. The signal from the reporter gene is usedas an indirect determinant for the translation of endogenous proteinsdriven from the same promoter.

Microplate Capture and Detection Assays use immobilized DNA probes tocapture specific protein-DNA interactions and confirm protein identitiesand relative amounts with target specific antibodies. Typically, a DNAprobe is immobilized on the surface of 96- or 384-well microplatescoated with streptavidin. A cellular extract is prepared and added toallow the binding protein to bind to the oligonucleotide. The extract isthen removed and each well is washed several times to removenon-specifically bound proteins. Finally, the protein is detected usinga specific antibody labelled for detection. This method can be extremelysensitive, detecting less than 0.2 pg of the target protein per well.This method may also be utilized for oligonucleotides labelled withother tags, such as primary amines that can be immobilized onmicroplates coated with an amine-reactive surface chemistry.

DNA Footprinting is one of the most widely used methods for obtainingdetailed information on the individual nucleotides in protein—DNAcomplexes, even inside living cells. In such an experiment, chemicals orenzymes are used to modify or digest the DNA molecules.• When sequencespecific proteins bind to DNA they can protect the binding sites frommodification or digestion. This can subsequently be visualized bydenaturing gel electrophoresis, where unprotected DNA is cleaved more orless at random. Therefore it appears as a ‘ladder’ of bands and thesites protected by proteins have no corresponding bands and look likefoot prints in the pattern of bands. The foot prints there by identifyspecific nucleosides at the protein—DNA binding sites.

Microscopic techniques include optical, fluorescence, electron, andatomic force microscopy (AFM).

Chromatin immunoprecipitation analysis (ChIP) causes proteins to bindcovalently to their DNA targets, after which they are unlinked andcharacterized separately.

Systematic Evolution of Ligands by EXponential enrichment (SELEX)exposes target proteins to a random library of oligonucleotides. Thosegenes that bind are separated and amplified by PCR.

Methods and compositions provided herein include, but are not limitedto, the following aspects.

Aspect 1: A synthetic composition comprising: (a) a guidepolynucleotide; (b) a Cas endonuclease comprising a C-terminal tri-splitRuvC domain further comprising a bridge helix and at least oneZinc-finger domain, an alpha helix bundle, and a plurality of betasheets forming a wedge-like domain, wherein the Cas endonuclease isfewer than 650 amino acids in length; and (c) a target sequencecomprising a nucleotide sequence that shares complementarity with theguide polynucleotide; wherein the guide polynucleotide and the Casendonuclease form a complex that cleaves a double stranded DNApolynucleotide comprising the target sequence.

Aspect 2: A synthetic composition comprising: (a) a guidepolynucleotide; (b) a Cas endonuclease derived from an organism of ataxonomy selected from the group consisting of: Archaea, Micrarchaeota,Acidibacillus sulfuroxidans, Candidatus Aureabacteria bacterium,Candidatus Micrarchaeota archaeon, Clostridium novyi, Parageobacillusthermoglucosidasius, Ruminococcus sp., and Syntrophomonas palmitatica;wherein the Cas endonuclease forms a complex with the guidepolynucleotide; and (c) a double-stranded DNA polynucleotide comprisinga target sequence that binds to the guide polynucleotide; wherein theguide polynucleotide and the Cas endonuclease form a complex thatcleaves the double stranded DNA polynucleotide comprising the targetsequence.

Aspect 3: The synthetic composition of Aspect 1 or Aspect 2, wherein theCas endonuclease further comprises a Zinc-finger domain near theN-terminus.

Aspect 4: The synthetic composition of Aspect 1 or Aspect 2, wherein thedouble-stranded DNA polynucleotide further comprises a PAM.

Aspect 5: The synthetic composition of Aspect 4, wherein the PAMcomprises a plurality of Thymine nucleotides.

Aspect 6: The synthetic composition of Aspect 1 or Aspect 2, furthercomprising a heterologous polynucleotide.

Aspect 7: The synthetic composition of Aspect 1 or Aspect 2, wherein theguide polynucleotide comprises a 20 nucleotide region of complementaritywith the target sequence.

Aspect 8: The synthetic composition of Aspect 1 or Aspect 2, wherein theguide polynucleotide is a duplex molecule comprising a tracrRNA and acrRNA.

Aspect 9: The synthetic composition of Aspect 1 or Aspect 2, wherein theguide polynucleotide is a single guide polynucleotide comprising a CasEndonuclease Recognition domain and a Variable Targeting domain.

Aspect 10: The synthetic composition of Aspect 6, wherein theheterologous polynucleotide is an expression element.

Aspect 11: The synthetic composition of Aspect 6, wherein theheterologous polynucleotide is a transgene.

Aspect 12: The synthetic composition of Aspect 6, wherein theheterologous polynucleotide is a donor DNA molecule.

Aspect 13: The synthetic composition of Aspect 6, wherein theheterologous polynucleotide is a polynucleotide modification template.

Aspect 14: The synthetic composition of Aspect 1 or Aspect 2, whereinthe CRISPR-Cas endonuclease further comprises a nuclear localizationsignal.

Aspect 15: The synthetic composition of Aspect 1 or Aspect 2, whereinthe CRISPR-Cas endonuclease is Cas-alpha, or a functional fragmentthereof.

Aspect 16: The synthetic composition of Aspect 1 or Aspect 2, whereinthe CRISPR-Cas endonuclease is a catalytically-inactive Cas-alpha.

Aspect 17: The synthetic composition of Aspect 1 or Aspect 2, whereinthe CRISPR-Cas endonuclease is a fusion protein comprising a functionalfragment of Cas-alpha.

Aspect 18: The synthetic composition of Aspect 17, wherein the fusionprotein further comprises another nuclease domain.

Aspect 19: The synthetic composition of Aspect 1 or Aspect 2, furthercomprising at least one additional polypeptide.

Aspect 20: The synthetic composition of Aspect 19, wherein theadditional polypeptide is selected from the group consisting of: Cas1,Cas2, and Cas4.

Aspect 21: The synthetic composition of Aspect 1 or Aspect 2, furthercomprising a cell.

Aspect 22: The synthetic composition of Aspect 21, wherein the cell is aeukaryotic cell.

Aspect 23: The synthetic composition of Aspect 21, wherein the cell is aplant cell.

Aspect 24: The synthetic composition of Aspect 23, wherein the plantcell is a monocot cell or a dicot cell.

Aspect 25: The synthetic composition of Aspect 23, wherein the plantcell is from an organism selected from the group consisting of: maize,soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye,barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa,sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

Aspect 26: The synthetic composition of Aspect 21, further comprising aguide polynucleotide comprising a variable targeting domain that issubstantially complementary to a target sequence in the genome of thecell

Aspect 27: A polynucleotide encoding the synthetic composition of Aspect1 or Aspect 2.

Aspect 28: The polynucleotide of Aspect 27, further comprising at leastone additional polynucleotide.

Aspect 29: The polynucleotide of Aspect 28, wherein the at least oneadditional polynucleotide is an expression element.

Aspect 30: The polynucleotide of Aspect 28, wherein the at least oneadditional polynucleotide is a gene.

Aspect 31: The synthetic composition of Aspect 30, wherein the gene isselected from the group consisting of: cas1, cas2, and cas4.

Aspect 32: The polynucleotide of Aspect 28, wherein at least onepolynucleotide is comprised within a recombinant construct.

Aspect 33: The synthetic composition of Aspect 1 or Aspect 2, wherein atleast one component is attached to a solid matrix.

Aspect 34: A synthetic composition comprising a target double-strandedDNA polynucleotide, a guide polynucleotide that is complementary to asequence in the double-stranded DNA polynucleotide, and a Casendonuclease at least 80% identical to a sequence selected from thegroup consisting of: SEQ ID NOs: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37,and 38, or a functional fragment or variant thereof.

Aspect 35: A synthetic composition comprising a target double-strandedDNA polynucleotide, a polynucleotide encoding a guide polynucleotidethat is complementary to a sequence in the double-stranded DNApolynucleotide, and a cas endonuclease gene at least 80% identical to asequence selected from the group consisting of: SEQ ID NOs: 13, 14, 15,16, 25, 26, 27, 28, 29, 30, and 31, or a functional fragment or variantthereof.

Aspect 36: A method for introducing a site-specific modification at atarget sequence in the genome of a cell, comprising: introducing intothe cell the synthetic composition from any of Aspects 1-35.

Aspect 37: A method of producing an organism with a modified genome,comprising: (a) introducing into at least one cell of the organism aheterologous composition comprising: i. a Cas-alpha endonuclease or acas-alpha polynucleotide encoding a Cas-alpha endonuclease, ii. a guidepolynucleotide comprising a variable targeting domain that issubstantially complementary to a target sequence in the genome of thecell, wherein the guide polynucleotide and Cas-alpha endonuclease arecapable of forming a complex that can recognize, bind to, and optionallynick or cleave the target sequence, iii. and a polynucleotidemodification template comprising at least one region that iscomplementary to a PAM sequence adjacent to a DNA target sequencerecognized by a Cas-alpha complex, wherein the at least one region thatcorresponds to a PAM sequence comprises at least one nucleotidemismatch; (b) incubating the cell, (c) generating a whole organism fromthe cell, and (d) verifying at least one nucleotide modification in thegenome of at least one cell of the organism as compared to the targetsequence of the genome of the cell prior to the introduction of theheterologous composition of (a).

Aspect 38: The method of Aspect 36 or 37, wherein the cell is aeukaryotic cell.

Aspect 39: The method of Aspect 38, wherein the eukaryotic cell isderived or obtained from an animal or a plant.

Aspect 40: The method of Aspects 39, wherein the plant is a monocot or adicot.

Aspect 41: The method of Aspects 39, wherein the plant is selected fromthe group consisting of: maize, soybean, cotton, wheat, canola, oilseedrape, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass,switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis,safflower, and tomato.

Aspect 42: The method of Aspect 36 or 37, further comprising introducinga heterologous polynucleotide.

Aspect 43: The method of Aspect 42, wherein the heterologouspolynucleotide is a donor DNA molecule.

Aspect 44: The method of Aspect 42, wherein the heterologouspolynucleotide is a polynucleotide modification template that comprisesa sequence at least 50% identical to a sequence in the cell.

Aspect 45: A progeny of the organism obtained by the method of Aspect37, wherein the progeny retains the at least one nucleotide modificationin at least one cell.

Aspect 46: A method of modifying a genomic sequence of a target cell,the method comprising providing a Cas endonuclease comprising an aminoacid sequence that is at least 95% to 100% identical to one of SEQ IDNOS: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, and 38 and a guidepolynucleotide that targets the genomic sequence of the target cell; andintroducing a double-strand break in the genomic sequence of the targetcell, thereby modifying the genomic sequence of the target cell.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention. For instance,while the particular examples below may illustrate the methods andembodiments described herein using a specific target site or targetorganism, the principles in these examples may be applied to any targetsite or target organism. Therefore, it will be appreciated that thescope of this invention is encompassed by the embodiments of theinventions recited herein and in the specification rather than thespecific examples that are exemplified below. All cited patents,applications, and publications referred to in this application areherein incorporated by reference in their entirety, for all purposes, tothe same extent as if each were individually and specificallyincorporated by reference.

EXAMPLES

The following are Examples of specific embodiments of some aspects ofthe invention. The Examples are offered for illustrative purposes only,and are not intended to limit the scope of the invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1: Identification and Characterization of Novel Class Cas-AlphaCRISPR-Cas Systems

In this Example, methods for identifying novel Class 2 CRISPR (ClusteredRegularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated)loci using identification of operon-like gene architecture and proteinstructural analyses are described.

First, arrays of CRISPRs were detected within microbial sequences usingPILER-CR (Edgar, R. (2007) BMC Bioinformatics, 8:18) and MinCED (Bland,C. et al. (2007) BMC Bioinformatics, 8:209) software programs. Next,known CRISPR-Cas systems were removed from the dataset by searching theproteins encoded in the vicinity (20 kb 5′ and 20 kb 3′ (wherepossible)) of the CRISPR array for homology with known CRISPR associated(Cas) proteins utilizing a set of position specific scoring matrices(PSSMs) encompassing all known Cas protein families as described inMakarova, K. et al. (2015) Nature Reviews Microbiology, 13:722-736. Toaid in the complete removal of known Class 2 CRISPR-Cas systems,multiple-sequence alignment of protein sequences from a collection ofothologs from each family of Class 2 CRISPR-Cas endonucleases (e.g.Cas9, Cpf1 (Cas12a), C2c1 (Cas12b), C2c2 (Cas13), C2c3 (Cas12c)) wasperformed using MUSCLE (Edgar R. (2004) Nucleic Acids Res.32:1792-1797). The alignments were examined, curated and used to buildprofile hidden Markov models (HMM) using HMMER (Eddy, S. R. (1998)Bioinformatics. 14:755-763; Eddy, S. R. (2011) PLoS Comp. Biol.,7:e1002195). The resulting HMM models were then utilized to furtheridentify and remove known Class 2 CRISPR-Cas systems from the dataset.Next, using PSSM specific searches as described above, the CRISPR locithat remained were evaluated for the presence of genes encoding proteinsimplicated as being important for spacer insertion and adaptation, Cas1and Cas2 (Makarova, K. et al. (2015) Nature Reviews Microbiology,13:722-736). CRISPR loci containing cas1 and cas2 genes were thenselected and further examined to determine the proximity, order anddirectionality of the undefined genes encoded in the locus relative tothe cas1 and cas2 genes and CRISPR array. Only those CRISPR loci formingan operon-like structure where a large (≥1500 bp open-reading frame)undefined gene was present close to and in the same transcriptionaldirection as the cas1 and cas2 genes were selected for further analysis.Next, the protein encoded in the undefined gene was analyzed forsequence and structural features indicative of a Class 2 endonucleasethat is capable of cleaving DNA. First, depending on how much similarityexisted between a candidate sequence and known proteins, variousbioinformatics tools were employed to reveal its conserved functionalfeatures, from pairwise comparison, to family profile search, tostructural threading, and to manually structural inspection. In general,homologous sequences for a new candidate protein were first collected bya PSI-BLAST (Altschul, S. F. et al. (1997) Nucleic Acids Res.25:3389-3402) search against the National Center for BiotechnologyInformation (NCBI) non-redundant (NR) protein collection with cut-offe-value of 0.01. After redundancy reduction at ˜90% identical level,groups of homologous sequences with various member inclusion thresholds(such as >60, 40, or 20% identity) were aligned to reveal conservedmotifs by multiple-sequence alignment tools, MSAPRobs (Liu, Y. et. al.(2010) Bioinformatics. 26:1958-1964) and Clustalw. The most conservedhomologous sequences underwent a sequence to family-profile search byHMMER (Eddy, S. R. (1998) Bioinformatics. 14: 755-763), against numerousdomain databases including Pfam, Superfamily, and, SCOP (Murzin, A. G.et al. (1995) J. Mol. Biol. 247:536-540) and home-built structure-basedprofiles. Separately, the resulting candidate's homologous sequencealignment was also used to generate a candidate protein profile withaddition of predicted secondary structures. The candidate profile wasfurther used to do a profile-profile search by HHSEARCH (Soding, J. etal. (2006) Nucleic Acids Res. 34:W374-378), against pdb70_hhm andPfam_hhm profile databases. In the next step, all detectedsequence-structure relationships and the conserved motifs were threadedinto a 3D structure template with MODELLER or manually mapped into theknown structural reference on Discovery Studio (BIOVIA) and Pymol(Schrodinger). Finally, to verify and confirm the potential biologicalrelevance as a Class 2 endonuclease, the catalytic or most conservedresidues and key structural integrity were manually inspected andevaluated in light of the protein's biochemical function. Followingstructural identification of the key features indicative of a Class 2endonuclease (e.g. DNA cleavage domain(s)), the other proteins encodedwithin the locus (5 kb 5′ and 5 kb 3′ from the ends of the newly definedCRISPR-Cas system (where possible)) were next examined for homology toknown proteins families using InterProScan software (EMBL-EBI, UK) andthrough comparison with the NCBI NR protein collection using the BLASTprogram (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410). Genesencoding proteins with similarity (at least 30% identity) to knownproteins were annotated as such in the CRISPR-Cas locus.

Initially, 4 novel Class 2 CRISPR-Cas system were identified fromunknown microbes (Table 1). As shown in FIGS. 1A and B, encoded in eachlocus was an intact CRISPR-Cas system comprising all the componentsrequired for acquisition and interference. These included genes thattogether encoded proteins needed for acquiring and integrating spacers(Cas1, Cas2, and optionally Cas4) and a novel protein comprising a DNAcleavage domain, Cas-alpha (α), in an operon-like structure adjacent toa CRISPR array (Table 1).

TABLE 1 Novel Class 2 CRISPR-Cas Systems Cas-alpha (α) 1, 2, 3, and 4Cas1 Cas2 Cas4 Casα SEQID NO Locus Name Organism SEQID SEQID SEQID GeneProtein SEQID NO Casα1 Candidatus Micrarchaeota archaeon 1 5 9 13 17 21Casα2 Candidatus Micrarchaeota archaeon 2 6 10 14 18 22 Casα3 CandidatusAureabacteria bacterium 3 7 11 15 19 23 Casα4 Uncultured Archaeon 4 8 1216 20 24

Next, comparisons of the Cas-alpha endonucleases with the NCBI NRprotein collection, using BLAST, followed by analysis with MinCED tofind proteins that were near (≤5 kb) a CRISPR array, produced 7additional CRISPR systems (Table 2). The locus gene architectureuncovered for these new proteins is shown in FIGS. 1C and 1D. The locusencoding Cas-alpha6 comprised intact cas1 and cas4 genes in addition toa partial cas1 gene (FIG. 1C) while Cas-alpha5, 7, 8, 9, 10, and 11contained only the endonuclease gene adjacent to the CRISPR array (FIG.1D). Loci for Cas-alphas 18 and 19 are depicted in FIG. 21A, with themechanism of action depicted in FIG. 21B.

TABLE 2a Cas-alpha (α) endonucleases 5-11 Locus SEQID NO. SEQID NameOrganism Gene Protein NO. Casα5 Candidatus Micrarchaeota archaeon 25 3239 Casα6 Uncultured Archaeon 26 33 40 Casα7 Parageobacillusthermoglucosidasius 27 34 41 Casα8 Acidibacillus sulfuroxidans 28 35 42Casα9 Ruminococcus sp. 29 36 43 Casα10 Syntrophomonas palmitatica 30 3744 Casα11 Clostridium novyi 31 38 45

Structural examination of these proteins revealed that they weredistinct from previously described Class 2 CRISPR-Cas endonucleasescapable of double stranded DNA target recognition and cleavage. First,the size (422-613 amino acids) of the endonuclease was remarkablycompact compared to other known Class 2 CRISPR-Cas systems. Second, thefirst amino (N)-terminal half of the protein was highly variable insequence composition as evident by the lack of conservation of even asingle amino acid (except a starting methionine). Despite this,secondary structure predictions (PSIPRED (Jones, J. T. (1999) J. Mol.Biol. 292:195-202)) indicated the presence of mixed beta-strands andalpha helices suggesting the presence of a Wedge-like (WED) oroligonucleotide binding domain (OBD) structure and Helical Bundle in theN-terminal region of all Cas-alpha proteins. In the carboxyl(C)-terminal half of the proteins, key catalytic residues and structurescomprising a tri-split RuvC domain were conserved (FIG. 2).Additionally, all proteins contained a bridge-helix domain and azinc-finger domain inserted between RuvC subdomains I-II andrespectively (FIG. 2). It should be noted that additionalzinc-finger-like motifs were detected in the Cas-alphas-1, 2, 3, 4, and10 proteins. For Cas-alphas-1, 2, 3, and 4 a second zinc-finger motifwas located near the N-terminus (e.g., amino acid positions 70-96 and63-111 in Cas-alpha-1 and 2, respectively) (FIGS. 8A-D) while forCas-alpha-10 two additional zinc-finger motifs were identified in theC-terminal half of the protein (FIG. 8J). Here, one of the extrazinc-finger domains was located in tandem with the first (Cas-alpha-10amino acid positions 376-422) between RuvC II and III sub-domains andthe third was found after RuvC sub-domain III (Cas-alpha-10 amino acidpositions 466-482) (FIG. 8J). Examples of the Cas-alpha sequences andmotifs recovered are shown in FIGS. 8A-K for Cas-alphas 1-11,respectively. FIG. 9 depicts how some of the Cas-alpha domains interactwith the hybrid duplex target DNA/guide RNA, using the Cas12b (C2c1)protein backbone (PDB:5wti) as a reference.

Sequence analysis of Cas-alphas 1 through 129, aligned with MUSCLEmultiple sequence alignment, revealed unique motifs that arecharacteristic for Cas-alpha endonucleases, relative to the amino acidposition numbers of SEQ ID NO:17 (Table: a Glycine (G) at position 337,a Glycine (G) at position 341, a Glutamic Acid (E) at position 430, aLeucine (L) at position 432, a Cysteine (C) at position 487, a Cysteine(C) at position 490, and/or a Cysteine (C) at position 507. A Cas-alphaendonuclease comprises the following motifs: GxxxG, ExL, Cx_(n)C, andCx_(n)(C,H) (wherein x_(n)=2-4 residues). A Cas-alpha endonucleasecomprises one or more zinc finger domains. Table 2b includes some of theconserved motifs found in Cas-alpha endonucleases.

TABLE 2b Cas-alpha (α) endonuclease conserved motifs Motifs aredescribed with the beginning amino acid (aa) position for each sequence(and end if >= 6 aa). x = any amino acid (n = any number, whereapplicable). G = Glycine, E = Glutamate, L = Leucine, C = Cysteine, H =Histidine. GxxxG ExL Cx_(n)C Cx_(n)(C,H) SEQID Cas- aa aa aa aa NO:alpha # Clade Length motif start motif start motif start motif start 171 1 544 GVDIG 337 ENL 430 CSSC 487 CLNPTC  507- 512 18 2 1 586 GIDRG 365EDL 471 CSKC 532 CLKC 550 19 3 1 603 GIDRG 380 EDL 481 CAYC 539 CKLH 56420 4 529 GIDVG 324 ENL 422 CSKC 475 CEKC 500 32 5 1 613 GIDRG 381 EDL482 CAYC 540 CLNPNC  565- 570 33 6 500 GLDVG 284 EDL 382 CSNPNC  435-CEKC 462 440 34 7 7 424 GVDLG 224 EDL 325 CSKC 372 CIEC 391 35 8 7 422GIDLG 223 EDL 324 CSEC 372 CRAC 391 36 9 6 440 GVDVG 229 EKL 330 CSKC378 CLEC 397 37 10 6 497 GVDLG 226 ENL 327 CSMC 376 CKQC 395 38 11 6 497GVDLG 290 ELL 390 CSKC 438 CKKC 457 254 12 6 461 GVDLG 271 EKL 371 CSRC418 CTKC 437 255 13 6 448 GIDLG 223 EDL 333 CNNC 382 CKVC 404 256 14 6430 GIDVG 216 ENL 328 CSLC 377 CVVC 399 257 15 6 436 GVDLG 229 EDL 330CSKC 378 CLKC 397 258 16 6 402 GVDLG 214 EQL 314 CSKC 361 CIKC 380 25917 6 493 GIDIG 230 ENL 386 CSRC 433 CVNDEC  455- 460 260 18 6 398 GVDLG205 EKL 305 CSKC 352 CKEC 371 261 19 6 433 GVDLG 231 EDL 332 CSKC 381CVKC 400 262 20 6 482 GVDLG 275 ELL 375 CSEC 423 CKRC 442 263 21 6 482GVDLG 275 ELL 375 CSEC 423 CKRC 442 264 22 6 424 GIDVG 223 EDL 324 CCKC423 CIDC 390 265 23 2 451 GIDLG 237 EDL 328 CSEC 371 CEKC 398 266 24 7451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 267 25 6 451 GIDLG 226 EDL 336CNNC 385 CKVC 407 268 26 6 437 GIDLG 225 EDL 335 CSKC 384 CTVC 403 26927 6 440 GVDVG 229 EKL 330 CSKC 378 CLEC 397 270 28 6 439 GVDVG 229 EKL330 CSKC 378 CLKC 397 271 29 6 402 GVDLG 214 EQL 314 CSKC 361 CIKC 380272 30 7 493 GLDLG 278 EYL 378 CSKC 426 CKSC 445 273 31 6 421 GVDMG 230EKL 330 CSEC 377 CLEC 396 274 32 6 421 GVDMG 230 EKL 330 CSEC 377 CLEC396 275 33 507 GIDVG 284 EDL 382 CSNPSC  435- CEKC 462 440 276 34 541GIDRG 347 EKL 437 CSHC 492 CNKC 514 277 35 537 GIDRG 342 EEL 432 CSHC487 CNKC 509 278 36 534 GIDRG 338 EKL 428 CSHC 483 CNKC 505 279 37 726GIDFG 402 EDL 509 CSKC 563 CEFC 602 280 38 777 GIDRG 448 ESL 549 CAKC609 CSVH 739 281 39 610 GIDRG 304 EKL 405 CAKC 465 CMKH 579 282 40 564GIDAG 351 EKL 454 CSHC 509 CGKC 537 283 41 610 GIDRG 401 EQL 492 CSHC547 CNKC 579 284 42 6 327 GVDLG 137 EKL 237 CSRC 284 CTKC 303 285 43 2364 GIDLG 150 EDL 241 CSEC 292 CEKC 311 286 44 6 366 GVDLG 156 ELL 256CSKC 304 CKKC 323 287 45 6 401 GVDMG 210 EKL 310 CSEC 357 CLEC 376 28846 6 401 GVDMG 210 EKL 310 CSEC 357 CLEC 376 289 47 6 401 GVDMG 210 EKL310 CSEC 357 CLEC 376 290 48 6 401 GVDMG 210 EKL 310 CSEC 357 CLEC 376291 49 6 401 GVDMG 210 EKL 310 CSEC 357 CLEC 376 292 50 6 404 GVDMG 213EKL 313 CSEC 360 CLEC 379 293 51 6 404 GVDMG 213 EKL 313 CSEC 360 CLEC379 294 52 6 404 GVDMG 213 EKL 313 CSEC 360 CLEC 379 295 53 6 404 GVDMG213 EKL 313 CSEC 360 CLEC 379 296 54 6 404 GVDMG 213 EKL 313 CSEC 360CLEC 379 297 55 6 407 GVDLG 219 EQL 319 CSKC 366 CIKC 385 298 56 6 421GVDMG 230 EKL 330 CSEC 377 CLEC 396 299 57 7 422 GVDLG 224 EDL 325 CSKC372 CKSC 391 300 58 7 424 GVDLG 224 EDL 325 CSKC 372 CIEC 391 301 59 7427 GVDLG 229 EDL 330 CSKC 379 CKGC 397 302 60 6 428 GVDLG 216 EDL 326CSIC 373 CINC 395 303 61 6 433 GVDLG 231 EDL 332 CSKC 381 CVKC 400 30462 7 438 GIDLG 224 ELL 319 CSQC 366 CKQC 385 305 63 6 439 GVDLG 228 EDL328 CSCC 378 CKNPEC  397- 402 306 64 6 440 GVDVG 229 EKL 330 CSKC 378CLKC 397 307 65 6 441 GIDLG 226 EDL 336 CSKC 385 CVIC 407 308 66 7 443GIDMG 223 EDL 324 CSEC 371 CQQC 390 309 67 6 444 GIDLG 226 ENL 336 CNRC385 CVVC 407 310 68 7 445 GIDLG 231 ELL 329 CSQC 373 CKQC 392 311 69 7445 GIDLG 231 ELL 326 CSQC 373 CKQC 392 312 70 7 445 GIDLG 231 ELL 326CSQC 373 CKQC 392 313 71 7 445 GIDLG 231 ELL 326 CSQC 373 CKQC 392 31472 7 447 GIDMG 246 EEL 348 CSEC 395 CLSC 417 315 73 6 447 GIDLG 224 EDL334 CSKC 383 CIIC 405 316 74 5 449 GIDLG 251 ESL 354 CSQC 400 CNKC 418317 75 7 450 GIDMG 230 EDL 331 CSEC 378 CQQC 397 318 76 7 450 GIDMG 230EDL 331 CSEC 378 CQQC 397 319 77 7 450 GIDMG 230 EDL 331 CSEC 378 CQQC397 320 78 6 450 GIDLG 229 EDL 339 CSKC 388 CKVC 410 321 79 7 451 GIDMG231 ELL 332 CSQC 379 CKQC 398 322 80 7 451 GIDMG 231 ELL 332 CSQC 379CKQC 398 323 81 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 324 82 7 451GIDLG 231 ELL 332 CSQC 379 CKQC 398 325 83 7 451 GIDMG 231 ELL 332 CSQC379 CKQC 398 326 84 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 327 85 7451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 328 86 7 451 GIDMG 231 ELL 332CSQC 379 CKQC 398 329 87 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 33088 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 331 89 7 451 GIDMG 231 ELL332 CSQC 379 CKQC 398 332 90 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398333 91 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 334 92 7 451 GIDMG 231ELL 332 CSQC 379 CKQC 398 335 93 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC398 336 94 7 451 GIDMG 231 ELL 332 CSQC 379 CKQC 398 337 95 7 453 GIDMG233 EDL 332 CSEC 381 CQQC 400 338 96 7 453 GIDMG 233 EDL 334 CSEC 381CQQC 400 339 97 7 453 GIDMG 233 EDL 334 CSEC 381 CQQC 400 340 98 7 453GIDMG 233 EDL 334 CSEC 381 CQQC 400 341 99 7 453 GIDMG 233 EDL 334 CSEC381 CQQC 400 342 100 7 453 GIDMG 233 ENL 334 CSEC 381 CQQC 400 343 101 4453 GVDLG 252 ESL 350 CHVC 406 CTNPEC  423- 428 344 102 4 453 GVDLG 252ESL 350 CHVC 406 CTNPEC  423- 428 345 103 6 461 GVDLG 271 EKL 371 CSRC418 CTKC 437 346 104 6 461 GVDLG 271 EKL 371 CSRC 418 CTKC 437 347 105 6461 GVDLG 271 EKL 371 CSRC 418 CTKC 437 348 106 6 461 GVDLG 271 EKL 371CSRC 418 CTKC 437 349 107 6 461 GVDLG 271 EKL 371 CSRC 418 CTKC 437 350108 6 461 GVDLG 271 EKL 371 CSRC 418 CTKC 437 351 109 6 461 GVDLG 271EKL 371 CSRC 418 CTKC 437 352 110 9 463 GIDIG 244 EKL 347 CSKC 395 CQVC419 353 111 8 464 GIDLG 228 EDL 329 CSRC 376 CREC 395 354 112 7 471GVDLG 265 EFL 393 CSKC 411 CKSC 430 355 113 4 471 GVDLG 268 ESL 367 CSYC423 CTNPQC  440- 445 356 114 6 477 GVDLG 286 EKL 386 CSKC 433 CKKC 452357 115 7 478 GVDLG 265 EFL 365 CSKC 413 CKSC 432 358 116 7 478 GVDLG265 EFL 365 CSKC 413 CKSC 432 359 117 6 482 GVDLG 275 ELL 375 CSEC 423CKRC 442 360 118 6 482 GVDLG 275 ELL 375 CSEC 423 CKRC 442 361 119 6 482GVDLG 275 ELL 375 CSEC 423 CKRC 442 362 120 2 486 GIDLG 256 ERL 382 CPSC429 CPEC 451 363 121 7 489 GVDLG 278 EEL 378 CSKC 426 CKNKEC  445- 450364 122 6 491 GVDLG 275 EYL 374 CHIC 433 CKAC 452 365 123 4 492 GVDLG289 ESL 388 CSYC 444 CTNPQC  461- 466 366 124 7 496 GVDLG 265 EFL 365CSKC 413 CKSC 432 367 125 7 496 GVDLG 265 EFL 365 CSKC 413 CKSC 432 368126 6 497 GVDLG 290 ELL 390 CSKC 438 CKKC 457 369 127 4 497 GVDLG 293ESL 392 CSYC 448 CTNPQC  465- 470 370 128 6 536 GVNLG 305 ENL 405 CSIC452 CKDPNC  471- 476 371 129 2 543 GVDLG 253 EDL 363 CPSC 412 CPVC 434

Example 2: Cas-Alpha Guide RNA Solutions

In this Example, methods for determining the guide RNA(s) that supportdouble stranded DNA target recognition and cleavage for a novel group ofclass 2 CRISPR (Clustered Regularly Interspaced Short PalindromicRepeat)-Cas (CRISPR associated) endonucleases, Cas-alpha, are described.

One method relies on computational prediction to determine the sRNA(s)needed to form a functional complex with a Cas-alpha endonuclease.Briefly, the CRISPR array may be utilized to generate CRISPR RNA(s)(crRNA(s)) accounting for both possible transcriptional directions ofthe CRISPR array and various configurations of the repeat and spacer(e.g. repeat:spacer, spacer:repeat or repeat:spacer:repeat) that may bepreferred by the endonuclease. Additionally, a trans-encoding CRISPRassociated RNA(s) (tracrRNA(s)) may be computationally identified in thelocus as described in Karvelis, T. et al. (2015) Genome Biology. 16:253.Briefly, an alignment may be performed between the CRISPR repeatconsensus sequence with the locus sequence using BLAST or manually byhand. Regions of homology (separate from the CRISPR array) may then beexamined by analyzing the possible transcriptional directions of theputative tracrRNA(s) for secondary structures and possible terminationsignals present in an RNA version of the sense and anti-sense genomicDNA sequences surrounding the anti-repeat. The tracrRNA(s) may then beduplexed with various crRNA predictions or engineered to form a chimericnon-natural single guide RNA(s) (sgRNA(s)). crRNA(s), tracrRNA(s) andsgRNA(s) may be synthesized (IDT equivalent) or T7 transcribed with theTranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific)or equivalent for further experimentation.

Another method is reliant on the sequencing of small RNAs (sRNA-seq)that are produced from the novel Class 2 CRISPR-Cas locus. This may beperformed similar to the method described in Zetsche, B. et al. (2015)Cell. 163:1-13. Briefly, the CRISPR-Cas locus is placed onto an E. coliplasmid DNA, subsequent cultures containing the plasmid borne CRISPR-Caslocus are harvested by centrifugation, total RNA extracted using TRIzolMax Bacterial Isolation Kit (Thermo Fisher Scientific), small RNAsisolated using the mirVana miRNA Isolation Kit (Thermo FisherScientific) and libraries prepared for sequencing using the TruSeq SmallRNA Library Prep Kit (Illumina). Expression of the locus can be boostedusing known E. coli promoters. Following sequencing on a MiSeqinstrument (Illumina) or equivalent, the resulting sequence data ismapped (Bowtie 2 software (Langmead, B. et al. (2012) Nat. Methods.9:357-359) or equivalent) back to the locus to determine thetranscriptional and maturation patterns of the sRNA(s) encoded in thelocus.

Another method is reliant on the sequencing of small RNAs (sRNA-seq)that are co-purified with the Cas-alpha protein from the novel Class 2CRISPR-Cas locus. This may be performed similar to the method describedin Sinkunas, T. et al. (2013) EMBO J. 32:385-394 except Illumina deepsequencing may be employed to determine the sequence of the small RNA(s)needed to direct double stranded DNA target recognition and cleavage.Briefly, the CRISPR-Cas locus is placed onto an E. coli plasmid DNA. TheCas-alpha gene in the locus can be modified to also encode a proteinpurification tag. For example but not limited to a histidine (His),streptavidin (Strep), and/or maltose binding protein (MBP) tag.Alternatively, a “solo” Cas-alpha expression cassette encoding a His,Strep, and/or MBP tagged version of the Cas-apha protein can beco-transformed with the plasmid borne locus. Next, the plasmid(s) aretransformed into E. coli (for example but not limited to Artic Express(DE3) (ThermoFisher Scientific) and then cultures are harvested bycentrifugation. The cells are then lysed and tagged Cas-alpha proteinpurified by chromatography. Finally, small RNAs bound to the Cas-alphaprotein are extracted using TRIzol Max Bacterial Isolation Kit (ThermoFisher Scientific) or other suitable method and processed as describedabove.

The crRNA, tracrRNA, and sgRNA solutions are listed in Table 3 forselect Cas-alpha systems described herein.

TABLE 3 Cas-alpha (α) guide RNA solutions Repeat crRNA Consensus (SEQ IDtracrRNA sgRNA Name (SEQ ID NO.) NO.) (SEQ ID NO.) (SEQ ID NO.) Casα1 4657 60, 61, 62, 63 69, 70, 71, 72 Casα2 47 58 64, 65, 66, 67, 73, 74, 75,76, 185, 186, 187 208, 209 Casα3 48 177 — — Casα4 49 59 68 77 Casα5 50178 — — Casα6 51 179 188, 189, 190, 211, 212, 213, 191 214 Casα7 52 180192, 193 215, 216, 217 Casα8 53 181 194, 195, 196 218, 219, 220, 221Casα9 54 182 197, 198 222, 223, 224 Casα10 55 183 199, 200, 201, 225,226, 227, 202, 203 228, 229 Casα11 56 184 204, 205, 206, 230, 231, 232,207 233, 234

Example 3: Bacterial Cas-Alpha Expression Plasmids

In this Example, plasmid DNA expression constructs are generated toexamine Cas-alpha double stranded DNA target recognition and cleavage ina heterologous host, E. coli.

First, the CRISPR array of a native Cas-alpha CRISPR-Cas locus (FIG. 1)(SEQ ID NO:21) encoding the first Cas-alpha endonuclease examine herein,Cas-alpha1 (SEQ ID NO:17, was modified. This was accomplished byreducing the number of CRISPR units (repeat (SEQ ID NO:46):spacer:repeat(SEQ ID NO:46)) to three. Next, the spacer sequence between the repeatswas replaced with a sequence (SEQ ID NO:78) capable of base pairing withthe anti-sense strand of a double stranded target sequence, T2, adjacentto a 7 bp region of randomization from the plasmid DNA PAM librarydescribed in Karvelis et al., 2015. The resulting “complete” CRISPR-Caslocus engineered to target T2 (SEQ ID NO:79) (FIG. 3), was thensynthesized (GenScript) directly into a low copy E. coli plasmid DNA(pACYC184, NEB) resulting in plasmid DNA R-225. It should be noted thatduring the synthesis process a single nucleotide polymorphism (SNP) wasintroduced into the casa1 gene but that the SNP (C to A bp at position1284 of the gene) was silent and did not alter the amino acidcomposition of Casa1. To enhance expression of the modified Cas-alphaCRISPR-Cas locus, it was also cloned into pETduet-1 (Millipore Sigma)modified to contain a single isopropyl β-D-1-thiogalactopyranoside(IPTG) inducible T7 promoter, resulting in plasmid DNA R-652. Next, toconfirm that double stranded DNA target cleavage activity requiredCasa1, its gene (SEQ ID NO:13) was removed from plasmid R-652 yieldingplasmid DNA R-658. To confirm the minimal components required for doublestrand DNA target recognition and cleavage, the adaptation genes (cas1,2, and 4) and the region 3′ of the modified CRISPR array were removedfrom R-652, producing a “minimal” locus (SEQ ID NO:80) (as exemplifiedin FIG. 3) expression plasmid resulting in plasmid R-657.

For other Cas-alpha endonucleases, a plasmid DNA expression cassetteencoding a “minimal” locus modified to target T2 (FIG. 3) (theequivalent of R-657 for Cas-alpha1) was synthesized (GenScript) intopETduet-1 to assay for dsDNA target recognition and cleavage.Additionally, a “solo” cas-alpha gene fused to the 3′ end of a sequenceencoding a histidine (HIS) tag (10X-HIS SEQ ID NO:81 or 6X-HIS SEQ IDNO:82), maltose binding protein (MBP) tag (SEQ ID NO:83), and tobaccoetch virus cleavage site (TEV) (SEQ ID NO:84) was constructed by methodsknown in the art (FIG. 3). Native cas-alpha gene sequences or E. colicodon optimized versions were utilized. For optimized genes, codonconditioning was carried out using E. coli codon tables, genes adjustedfor ideal GC content, and repetitive sequences and gene destabilizingfeatures removed where possible. Finally, the tagged “solo” cas-alphagenes were cloned into either a Tetracycline (TET), IPTG, or arabinoseinducible plasmid DNA expression cassettes by methods known in the art.

Example 4: Cas-Alpha Protein Expression and Purification

In this Example, a method to recombinantly expression and purifyCas-alpha endonucleases are described.

Cas-alpha protein was expressed and purified using a tagged “solo”protein expression plasmid as described in Example 3. First, theexpression construct was transformed into either E. coli BL21(DE3) orArcticExpress (DE3) strains and cultures were grown in LB brothsupplemented with selective agent (e.g. ampicillin (100 μg/ml)). Afterculturing to an OD₆₀₀ of 0.5, temperature was decreased to 16° C. andexpression induced with IPTG (0.5 mM) or arabinose (0.2% (w/v)). After16 h, cells were pelleted and re-suspended in loading buffer (20Tris-HCl, pH 8.0 at 25° C., 1.5 M NaCl, 5 mM 2-mercaptoethanol, 10 mMimidazole, 2 mM PMSF, 5% (v/v) glycerol) and disrupted by sonication.Cell debris was removed by centrifugation. The supernatant was loaded onthe Ni²⁺-charged HiTrap chelating HP column (GE Healthcare) and elutedwith a linear gradient of increasing imidazole concentration (from 10 to500 mM) in 20 Tris-HCl, pH 8.0 at 25° C., 0.5 M NaCl, 5 mM2-mercaptoethanol buffer. The fractions containing Cas-alpha were pooledand subsequently loaded on a HiTrap heparin HP column (GE Healthcare)for elution using a linear gradient of increasing NaCl concentration(from 0.1 to 1.5 M). The next fractions containing Cas-alpha proteinwere pooled and the tag was cleaved by overnight incubation with TEVprotease at 4° C. To remove cleaved His-MBP-tag and TEV protease,reaction mixtures were loaded onto a HiTrap heparin HP 5 column (GEHealthcare) for elution using a linear gradient of increasing NaClconcentration (from 0.1 to 1.5 M). Next, the elution from the HiTrapcolumns was loaded on a MBPTrap column (GE Healthcare) and Cas-alphaprotein was collected as flow though. The collected fractions were thendialyzed against 20 mM Tris-HCl, pH 8.0 at 25° C., 500 mM NaCl, 2 mMDTT, and 50% (v/v) glycerol and stored at −20° C.

Example 5: Methods to Detect Cas-Alpha Double Stranded DNA TargetRecognition and Cleavage

In this Example, methods to detect double strand DNA target recognitionand cleavage by Cas-alpha endonucleases are described.

Lysate Assay

The detection of double stranded DNA target recognition and cleavage wascarried-out using cell lysates expressing a Cas-alpha endonuclease asshown in FIG. 3. First, a plasmid DNA encoding a Cas-alpha endonucleaseeither by itself or as part of a Cas-alpha CRISPR-Cas locus modified totarget the T2 sequence (see Example 3) was transformed into E. colicells (e.g., DH5α (Thermo Fisher Scientific), ArcticExpress (DE3)(Agilent Technologies), or NEB Stable (NEB)) by methods known in theart. Next, cell cultures carrying the gene encoding the Cas-alphaendonuclease were cultured to an optical density (OD) of 0.5 (using awavelength of 600 nm) in Luria broth (LB) media containing a suitableantibiotic (e.g., ampicillin) (FIG. 3 Step I). For plasmids thatrequired an inducing agent to stimulate expression (e.g., R-652), thetemperature was decreased to 16° C., and expression initiated withinducing agent (e.g., 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG))for 16 h. If no induction was required, cells were immediately harvestedafter reaching an OD₆₀₀ of 0.5. Next, cells were pelleted bycentrifugation (at 3,000 g for 5 min at 4° C.), media poured-off, andresuspended in 1 ml of lysis buffer (20 mM phosphate, pH 7.0, 0.5 MNaCl, 5% (v/v) glycerol) supplemented with 10 μl PMSF and transferred toice. The cells were then disrupted by sonication for 2 min (6 s pulsefollowed by 3 s pause) and cell debris removed by centrifugation at14,000 g for 30 min at 4° C. Next, for Cas-alpha proteins expressed as asolo component, 20 μl of supernatant containing the soluble Cas-alphaprotein was immediately combined with 2 μg of the T7 transcribed guideRNA(s) in the presence of 1 μl (40 U) of RiboLock RNase Inhibitor(Thermo Fisher Scientific) and incubated for 15 min at room temperature(FIG. 3 Step II). If the Cas-alpha endonuclease and guide RNAs wereexpressed together from the plasmid borne CRISPR-Cas locus, theclarified lysate containing Cas-alpha guide RNA ribonucleoproteincomplexes was not processed any further but used directly in the nextstep (FIG. 3 Step II). Digestion of a randomized PAM library was thenperformed by gently combining 10 μl of the Cas-alpha guide RNA lysatemixture with 90 μl of reaction buffer (10 mM Tris-HCl, pH 7.5 at 37° C.,100 mM NaCl and 1 mM DTT, 10 mM MgCl2) and 1 μg of the 7 bp randomizedPAM library from Karvelis et al. 2015 containing a T2 target sequence(FIG. 3 Step III). Alternatively, if the PAM sequence was known, 10 μlof the Cas-alpha guide RNA lysate mixture was combined with 1 μg ofplasmid DNA containing a fixed target sequence. After 1 h at 37° C.,reactions were subject to DNA end-repaired by incubating them with 1 μl(5U) of T4 DNA polymerase and 1 μl of 10 mM dNTP mix (Thermo FisherScientific) for 20 min at 11° C. The reaction was then inactivated byheating it to 75° C. for 10 min. To efficiently capture free DNA ends byadapter ligation, a 3′-dA overhang was added by incubating the reactionmixture with 1 μl (5 U) of DreamTaq polymerase (Thermo FisherScientific, EP0701) for 30 min at 72° C. Excess RNA was then removedfrom the reaction by incubating 1 μl of RNase A/T1 (Thermo FisherScientific) for 30 min at 37° C. The resulting DNA was then purifiedusing a GeneJet PCR Purification Kit (Thermo Fisher Scientific).

Next, an adapter with a 3′-dT overhang was prepared by annealing A1(5′-CGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′ (SEQ ID NO:85)) andphosphorylated A2 (5′-GATCGGAAGAGCGGTTCAGCAGGAATGCCG-3′ (SEQ ID NO:86))oligonucleotides by heating an equimolar mixture of the two for 5 min at95° C. and slowly cooling (˜0.1° C./s) to room temperature in Annealing(A) buffer (10 mM Tris-HCl, pH 7.5 at 37° C., 50 mM NaCl). The adapterwas then ligated to the end repaired 3′-dA overhanging cleavage productsby combining 100 ng of it and the adapter with 5 U of T4 Ligase (ThermoFisher Scientific) in 25 μl of ligation buffer (40 mM Tris-HCl, pH 7.8at 25° C., 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP, 5% (w/v) PEG 4000) andallowing the reaction to proceed for 1 h at room temperature (FIG. 3Step IV).

Next, the cleaved products containing the PAM sequence were enrichedusing R0 (5′-GCCAGGGTTTTCCCAGTCACGA-3′ (SEQ ID NO:87)) and the A1oligonucleotide specific to the 7 bp PAM library and adapter,respectively (FIG. 3 Step V). PCR was performed with PhusionHigh-Fidelity PCR Master Mix with high fidelity (HF) Buffer (ThermoFisher Scientific) using 10 μl of the ligation reaction as template. Atwo-step amplification protocol (98° C.—30 s initial denaturation, 98°C.—15 s, 72° C.—30 s denaturation, annealing and synthesis for 15 cyclesand 72° C.—5 min for final extension) was used. For the samplesassembled in the absence of a Cas-alpha, PCR was performed using the R0and the C0 primer (5′-GAAATTCTAAACGCTAAAGAGGAAGAGG-3′ (SEQ ID NO:88))pair with C0 being complementary to protospacer sequence. Next, theamplification products (148 bp and 145 bp for A1/R0 and C0/R0 primerpairs, respectively) were purified using a GeneJet PCR Purification Kit(Thermo Fisher Scientific).

Next, the sequences and indexes required for Illumina deep sequencingwere incorporated onto the ends of the Cas-alpha cleaved DNA fragmentsand the resulting products deep sequenced (FIG. 3 Step VI). This wasaccomplished through two rounds of PCR using Phusion High-Fidelity PCRMaster Mix in HF buffer (New England Biolabs) per the manufacturer'sinstruction. The primary PCR was assembled using 20 ng of Cas-alphacleaved adapter ligated PAM-sided template and allowed to proceed for 10cycles. The reaction uses a forward primer, F1(5′-CTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGCGGC-ATTCCTGCTGAAC-3′ (SEQ IDNO:89)) that can hybridize to the adapter and a reverse primer, R1(5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTCGGCGACGTTGGGTC-3′ (SEQ IDNO:90)), that binds to a site 3′ of the region of PAM randomization. Inaddition to hybridizing to the adapter ligated PAM fragment, the primersalso contain Illumina sequences extending off their 5′ ends. For theforward primer, the extra sequence includes a portion of the sequencerequired for bridge amplification(5′-CTACACTCTTTCCCTACACGACGC-TCTTCCGATCT-3′ (SEQ ID NO:91)) followed byan interchangeable unique index sequence (5′-AAGG-3′) that permitsmultiple amplicons to be deconvoluted if sequenced simultaneously. Forthe reverse primer, the additional sequence is comprised only of thatrequired for bridge amplification at the 3′ end of the amplicon(5′-CAAGCAGAAGACGGCATACGAGCTC-TTCCGATCT-3′ (SEQ ID NO:92)). Thefollowing PCR cycling conditions were used: 95° C.—30 s initialdenaturation, 95° C.—10 s, 60° C.—15 s, 72° C.—5 s denaturation,annealing and synthesis for 10 cycles and 72° C.—5 min for finalextension. Following primary PCR, a second round of PCR amplificationwas performed using 2 μl (in total volume of 50 μl) of the first roundPCR as template. The forward primer, F2(5′-AATGATACGGCGACCACCGAGATCTACACTCTTT-CCCTACACG-3′ (SEQ ID NO:93)),used in the secondary PCR hybridizes to the 5′ region of F1 furtherextending the sequences required for Illumina deep sequencing. Thereverse primer, R2 (5′-CAAGCAGAAGACGGCATA-3′ (SEQ ID NO:94)), used inthe secondary PCR simply binds to the 3′ end of the primary PCRamplicon. The following PCR cycling conditions were used: 95° C.—30 sinitial denaturation, 95° C. —10 s, 58° C.—15 s, 72° C.—5 sdenaturation, annealing and synthesis for 10 cycles and 72° C.—5 min forfinal extension. Following library creation, amplifications werepurified with a QIAquick PCR Purification Kit (Qiagen) per themanufacturer's instruction and combined into a single sample in anequimolar concentration. Next, the libraries were single-read deepsequenced on a MiSeq Personal Sequencer (Illumina) with a 25% (v/v)spike of PhiX control v3 (Illumina) and sequences post-processed anddeconvoluted per the manufacture's instruction. Note the original PAMlibrary was also sequenced as a control to account for inherent biasthat would affect downstream PAM analyses. This is carried out asdescribed above except the forward primer in the primary PCR, C1(5′-CTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAATAAACGCTAAAGAGGAAG AGG-3′(SEQ ID NO:95)), is used instead of F1 as it hybridizes directly to theprotospacer region in the uncut PAM library.

Next, evidence of double stranded DNA target recognition was evaluatedby searching for the presence of a PAM in the Cas-alpha cleavedfragments. This was accomplished by first generating a collection ofsequences representing all possible outcomes of double stranded DNAcleavage and adapter ligation within the target region. For example,cleavage and adapter ligation at just after the 21^(st) position of thetarget would produce the following sequence(5′-CCGCTCTTCCGATCTGCCGGCGACGTTGGGTCAACT-3′ (SEQ ID NO:96)) where theadapter and target sequences comprise 5′-CCGCTCTTCCGATCT-3′ (SEQ IDNO:97) and 5′-GCCGGCGACGTTGGGTCAACT-3′ (SEQ ID NO:98), respectively.Next, these sequences were searched for in the sequence datasets alongwith a 10 bp sequence 5′ of the 7 bp PAM region (5′-TGTCCTCTTC-3′ (SEQID NO:99)). Once identified, the intervening PAM sequence was isolatedby trimming away the 5′ and 3′ flanking sequences. Next, the frequencyof the extracted PAM sequences was normalized to the original PAMlibrary to account for bias inherent to the initial library. First,identical PAM sequences were enumerated, and frequency calculated versusthe total reads in the dataset. Then, normalization was performed foreach PAM using the following equation such that PAM sequences that wereunder- or over-represented in the initially library were accounted for:

Normalized Frequency=(Treatment Frequency)/(((ControlFrequency)/(Average Control Frequency)))

After normalization, a position frequency matrix (PFM) was calculated.This was done by weighting each nucleotide at each position based on thefrequency (normalized) associated with each PAM. For example, if a PAMof 5′-CGGTAGC-3′ had a normalized frequency of 0.15%, then the C atfirst position would be given a frequency of 0.15% when determining thenucleotide frequency for the first PAM position. Next, the overallcontribution of each nucleotide at each position in the dataset wassummed and organized into a table with the most abundant nucleotidesindicating Cas-alpha PAM preferences.

Evidence for Cas-alpha double stranded DNA target cleavage was evaluatedby examining the unique junction generated by Cas-alpha target cleavageand adapter ligation. First, a collection of sequences representing allpossible outcomes of double stranded DNA cleavage and adapter ligationwithin the T2 target region were generated (as detailed above). Next,the frequency of the resulting sequences was examined in each Illuminasequence dataset relative to negative controls (experiments setupwithout Cas-alpha). Protospacer-adapter ligation positions whereIllumina sequences were recovered in excess resulting in a peak or spikeof read coverage over negative controls were considered as evidence oftargeted DNA cleavage.

Example 6: Cas-Alpha Double Stranded DNA Target Recognition and Cleavage

In this Example, the molecular features that impart Cas-alpha doublestranded DNA target recognition and cleavage are identified.

Cas-Alpha is a PAM-Dependent dsDNA Endonuclease

Cas-alpha CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)-Cas (CRISPR associated) endonucleases have been reported toonly cleave single stranded DNA targets without the requirement of aprotospacer adjacent motif (PAM) (Harrington, L. B. et al. (2018)Science. 10.1126/science.aav4294). In this example, we provide evidencethat this novel group of CRISPR-Cas endonucleases, 1) requires a PAM incombination with a 2) guide RNA to 3) recognize and cleave a doublestranded DNA target site.

As shown in Table 4, PAM preferences were recovered for Cas-alpha1 whenusing plasmid R-225 (containing a fully intact Cas-alpha CRISPR-Caslocus modified to target the T2 sequence) providing the first evidenceof Cas-alpha double stranded DNA target recognition. PAM preferencesonly occurred when assuming target DNA cleavage and adapter ligation ata position 21 bp 3′ of the PAM region. To confirm double stranded DNAcleavage activity, a plasmid DNA was constructed containing a fixeddouble stranded DNA target sequence (SEQ ID NO:100) comprised of anon-randomized PAM (5′-TTAT-3′) immediately 5′ of the T2 target sequence(SEQ ID NO:101). Then using plasmids R-225 and R-654 (see Example 3) andthe fixed target sequence, experiments were repeated. As shown in FIGS.4A-E, these experiments resulted in a spike of sequence reads recoveredat the aforementioned position relative to the negative control. ForR-654, a T7 IPTG inducible promoter enhanced the fraction of readsrecovered (approaching nearly 40% of all reads) immediately after the21⁴ position downstream of the PAM.

TABLE 4 Protospacer adjacent motif (PAM) preferences for Cas-alpha1Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 22.95% 22.92% 16.12%  2.74% 1.83%  5.71%  1.59% T 24.97% 35.45% 37.93% [90.53%] [95.44%]  1.24%[96.44%] A 25.74% 26.48% 30.92%  2.64%  1.06% [64.16%]  1.06% G 26.34%15.15% 15.03%  4.09%  1.66% /28.89%/  0.91% Consensus N N N T T A T

To confirm that the observed double stranded DNA target recognition andcleavage activity observed was the result of only Cas-alpha1, thetracrRNA encoding region, and the modified CRISPR array, experimentswere conducted with plasmid (R-657) containing a minimal Cas-alphaCRISPR-Cas locus (comprised only of the Cas-alpha gene, the regionencoding the tracrRNA, and the modified CRISPR array) and the fixeddouble stranded DNA target sequence. As illustrated in FIG. 4D, asimilar cleavage signature was recovered at the 21^(st) position 3′ ofthe PAM. Finally, to verify that Cas-alpha was required for the observedcleavage activity, experiments were also conducted when the Cas-alphagene was removed from the CRISPR-Cas locus (R-658). As shown in FIG. 4E,no DNA cleavage activity was detected. Taken together, this provides thefirst evidence for Cas-alpha double stranded DNA target cleavage.

Double stranded DNA target recognition and cleavage was examined for asecond Cas-alpha protein, Cas-alpha4 (SEQ ID NO:20). Using a soloCas-alpha4 expression cassette (see Example 2 and FIG. 3), T7transcribed guide RNA(s) targeting T2, a sequence adjacent to the 7 bprandomized PAM library described in Karvelis et al., 2015, were combinedwith E. coli lysate containing Cas-alpha4 expressed protein. Todetermine the orientation of PAM recognition relative to spacerrecognition, guide RNA(s) were designed to base pair with either thesense or anti-sense strands of the T2 target (Table 5) (FIG. 5). If theguide RNA(s) designed to base pair with the sense strand result in therecovery of PAM preferences and yield a cleavage signal, then theprotospacer is on the anti-sense strand and PAM recognition occurs 3′relative to it (FIG. 5A). Conversely, if the guide RNA(s) designed tobase pair with the anti-sense strand produce PAM preferences and acleavage signal, then the protospacer is on the sense strand and PAMrecognition occurs in an orientation 5′ to it (FIG. 5B). Upon evaluationof the frequency of adapter ligation at each position in the T2protospacer target, a peak comprising nearly 30% of all reads wasrecovered just after the 24th bp 3′ of the PAM (FIGS. 6C and 6E). Bothguide RNAs producing cleavage signal were designed to target theanti-sense strand of the protospacer, thus, indicating that PAMrecognition occurs 5′ of the protospacer. Next, PAM recognition wasevaluated for Cas-alpha4. As shown in Tables 6 and 7, T-rich PAMpreferences similar to Cas-alpha1 were also recovered for Cas-alpha2when the guide RNAs, T2-2 sgRNA or T2-2 crRNA/tracrRNA, were used.

TABLE 5 Cas-alpha4 T7 transcribed guide RNAs Single crRNA tracrRNA T2Strand Guide RNA SEQID SEQID Name Targeted SEQID NO NO NO T2-1 sgRNASense 102 — — T2-2 sgRNA Anti-Sense 103 — — T2-1 crRNA/tracrRNA Sense —104 68 T2-2 crRNA/tracrRNA Anti-Sense — 105 68

TABLE 6 Protospacer adjacent motif (PAM) preferences for Cas-alpha4 whenpaired with the guide RNA, T2-2 sgRNA Displayed as a position frequencymatrix (PFM). PAM positions are numbered backward from the firstposition of the protospacer target with position −1 being immediately 5′of the first position of the T2 protospacer target. Numbers in brackets[x] represent strong PAM preferences, numbers in slashes /x/ representweak PAM preferences. PAM Position −7 −6 −5 −4 −3 −2 −1 Nucleotide C27.81% 21.82% 19.53%  3.37%  2.53%  5.80%  0.59% T 20.37% 32.63% 32.81%[92.55%] [97.41%] [87.54%]  8.18% A 21.98% 31.52% 28.51%  2.65%  0.01% 3.05% [45.46%] G 29.85% 14.02% 19.14%  1.43%  0.06%  3.61% [45.77%]Consensus N N N T T T R

TABLE 7 Protospacer adjacent motif (PAM) preferences for Cas-alpha4 whenpaired with the guide RNA, T2-2 crRNA/tracrRNA Displayed as a positionfrequency matrix (PFM). PAM positions are numbered backward from thefirst position of the protospacer target with position −1 beingimmediately 5′ of the first position of the T2 protospacer target.Numbers in brackets [x] represent strong PAM preferences, numbers inslashes /x/ represent weak PAM preferences. PAM Position −7 −6 −5 −4 −3−2 −1 Nucleotide C 28.66% 20.32% 18.37%  2.79%  1.78%  4.79%  0.62% T19.41% 33.80% 33.84% [94.11%] [97.97%] [89.27%]  7.33% A 20.56% 33.10%28.94%  2.14%  0.01%  2.83% [46.66%] G 31.36% 12.78% 18.85%  0.95% 0.24%  3.12% [45.39%] Consensus N N N T T T R

To confirm our findings in an entirely biochemical environment,double-stranded DNA target cleavage was reconstituted in vitro. This wasaccomplished by using purified Cas-alpha4 protein (Example 4) and invitro T7 transcribed single guide RNA (sgRNA) (SEQ ID NO: 77) (Example2) to digest double stranded DNA targets. First, to formribonucleoprotein (RNP) complexes, a 1:1 molar ratio of Cas-alpha4 andsgRNA were incubated in complex assembly buffer (10 mM Tris-HCl, pH 7.5at 37° C., 100 mM NaCl, 1 mM EDTA, 1 mM DTT) at 37° C. for 30 min. 100nM of the resulting RNP was then combined with 3 nM of eithersupercoiled (SC) or linearized plasmid DNA containing a sgRNA targetsequence flanked by a Cas-alpha4 PAM (5′-TTTA-3′) in reaction buffer(2.5 mM Tris-HCl, pH 7.5 at 37° C., 25 mM NaCl, 0.25 mM DTT and 10 mMMgCl2) and incubated for 30 min. at 37° C. Then, reactions were stoppedand analyzed by non-denaturing agarose gel electrophoresis and ethidiumbromide staining. As shown in FIG. 15A, SC plasmid DNA was completelyconverted to a linear form (FLL), thus, illustrating the formation of adsDNA break. Additionally, cleavage of linear DNA resulted in DNAfragments of an expected size further validating Cas-alpha4 mediateddsDNA break formation (FIG. 15A). Next, by excluding either the PAM orthe sgRNA target, we confirmed that Cas-alpha4 absolutely requires a PAMand guide RNA to cleave a dsDNA target (FIG. 15B).

The type of dsDNA break generated by Cas-alpha4 was examined next. Usingrun-off sequencing, we observed that Cas-alpha4 generates 5′ staggeredoverhanging DNA cut-sites. Cleavage predominantly occurred centeredaround positions 20-24 bp in respect to PAM-sequence (FIG. 15C).

Next, we investigated if Cas-alpha4 induces non-specific ssDNAdegradation activity following dsDNA target recognition. Here, reactionswere assembled as described above except 100 nM of dsDNA containing a 5′PAM and adjacent sgRNA target was used as an activator and 100 nM of M13single-stranded DNA was included to detect Cas-alpha4 induced ssDNaseactivity. Reactions were also setup without the sgRNA to illustrate thatdsDNA targeting is a prerequisite of indiscriminate ssDNA cleavage. Asshown in FIG. 15D, trans-acting ssDNase activity of Cas-alpha4 wasactivated by dsDNA only in the presence of a guide RNA

To investigate the broad applicability of our findings, Cas-alphas 2(SEQ ID NO:18), 3 (SEQ ID NO:19), 5 (SEQ ID NO:32), 6 (SEQ ID NO:33), 7(SEQ ID NO:34), 8 (SEQ ID NO:35), 9 (SEQ ID NO:36), 10 (SEQ ID NO:37),and 11 (SEQ ID NO:38) were also evaluated for double stranded DNA targetrecognition and cleavage. Using a minimal CRISPR-Cas locus (comprisingthe cas-alpha endonuclease gene, the region encoding the tracrRNA, andthe T2 modified CRISPR array (FIG. 3)) synthesized into a bacterial T7expression cassette (pETduet-1 (MilliporeSigma)), E. coli lysateexperiments were performed as described in Example 4 with and withoutIPTG induction. As shown in FIGS. 16A-16T, double stranded DNA targetcleavage was detected for all except Cas-alpha 5. In general, andsimilar to results with Cas-alpha 1 and 4, protospacer positions 21 and24 3′ of the region of PAM randomization exhibited the highest frequencyof adapter-ligated reads. Similar to Cas-alpha 1 and 4, 5′ PAMrecognition was also recovered (Tables 8-15).

Taken together, the data described herein provides evidence thatCas-alpha proteins are directed by guide RNA(s) to recognize and cleavedouble stranded DNA target sites in the presence of a 5′ PAM.

TABLE 8 Protospacer adjacent motif (PAM) preferences for Cas-alpha2Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 27.16% 27.49% 24.86% 18.78% 1.04%  0.94%  1.59% T 23.45% 27.16% 30.20% /46.93%/ [98.67%] [99.03%] 0.91% A 23.83% 27.04% 22.36% 13.40%  0.20%  0.00% [44.59%] G 25.56%18.31% 22.58% 20.89%  0.09%  0.03% [52.91%] Consensus N N N N (T > V) TT R

TABLE 9 Protospacer adjacent motif (PAM) preferences for Cas-alpha3Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 24.20% 22.03% 12.82%  3.44% 0.03%  2.29%  0.49% T 24.96% 28.54% /41.83%/ [81.31%] [99.83%] [97.50%] 1.67% A 28.67% 34.66% 34.60%  7.52%  0.01%  0.02% [42.66%] G 22.17%14.76% 10.75%  7.72%  0.13%  0.19% [55.18%] Consensus N N N (W > S) T TT R

TABLE 10 Protospacer adjacent motif (PAM) preferences for Cas-alpha6Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 25.57% 23.71% 20.38%  6.56% 0.09%  0.75%  1.72% T 20.76% 24.38% 33.64% [88.29%] [99.70%] [98.29%] 0.50% A 25.73% 30.31% 31.04%  3.36%  0.04%  0.90% [55.68%] G 27.94%21.60% 14.94%  1.79%  0.18%  0.06% [42.10%] Consensus N N N T T T R

TABLE 11 Protospacer adjacent motif (PAM) preferences for Cas-alpha7Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 23.92% 14.67% 30.89% 35.82% 4.62%  3.77%  6.56% T 27.66% 35.68% /40.15%/ /45.01%/ [94.62%] [92.32%]11.97% A 23.41% 38.99% 17.44% 13.91%  0.54%  2.48% /53.85%/ G 25.00%10.66% 11.53%  5.26%  0.22%  1.43% 27.62% Consensus N N N (Y > R) N (Y >R) T T N (A > G > Y)

TABLE 12 Protospacer adjacent motif (PAM) preferences for Cas-alpha8Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 23.87% 15.70% 32.93% 33.72% 0.09%  0.31%  2.09% T 34.05% /47.29%/ /40.50%/ [59.74%] [99.80%][99.48%]  6.62% A 17.43% 30.27% 14.82%  6.06%  0.00%  0.00% [46.71%] G24.65% 6.74% 11.76%  0.48%  0.11%  0.20% [44.58%] Consensus N N (W > S)N (Y > R) T T T R

TABLE 13 Protospacer adjacent motif (PAM) preferences for Cas-alpha9Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 27.75% 24.68% 21.31% 27.66%[94.23%]  0.61% 12.51% T 25.91% 27.08% 28.18% 25.92%  4.72% [97.50%][87.48%] A 18.82% 29.30% 28.38% 23.24%  0.86%  0.18%  0.00% G 27.52%18.95% 22.12% 23.17%  0.18%  1.72%  0.00% Consensus N N N N C T T

TABLE 14 Protospacer adjacent motif (PAM) preferences for Cas-alpha10Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 28.95% 22.41% 29.44% 11.54% 5.78%  4.62% [74.42%] T 26.49% 27.58% 20.35% 23.90% [84.34%] [93.40%]20.79% A 16.96% 34.00% 22.28% 20.71%  6.54%  0.97%  3.16% G 27.60%16.01% 27.93% /43.85%/  3.35%  1.01%  1.64% Consensus N N N N T T C (T >W > C)

TABLE 15 Protospacer adjacent motif (PAM) preferences for Cas-alpha11Displayed as a position frequency matrix (PFM). PAM positions arenumbered backward from the first position of the protospacer target withposition −1 being immediately 5′ of the first position of the T2protospacer target. Numbers in brackets [x] represent strong PAMpreferences, numbers in slashes /x/ represent weak PAM preferences. PAMPosition −7 −6 −5 −4 −3 −2 −1 Nucleotide C 24.79% 23.95% 27.18% 26.09%[100.00%] [99.92%]  1.66% T 27.18% 26.39% 25.05% 25.23%  0.00%  0.01%[29.92%] A 23.12% 24.69% 23.35% 25.36%  0.00%  0.07% [33.27%] G 24.91%24.97% 24.42% 23.33%  0.00%  0.00% [35.15%] Consensus N N N N C C D

Determination of Optimal Conditions for Cas-Alpha Cleavage

Biochemical experiments to determine parameters and conditions foroptimal RNA-guided Cas-alpha endonuclease cleavage of dsDNA wereconducted using methods known in the art. Briefly, purified Cas-alphaprotein and T7 transcribed guide RNA were incubated in Complex Assembly(CA) buffer (10 mM Tris-HCl, pH 7.5 at 37° C., 100 mM NaCl and 1 mMDTT). The resulting RNP complexes were then combined with doublestranded plasmid DNA containing a 5′ PAM immediately adjacent to aregion with complementarity to the guide RNA (for example, asillustrated in FIG. 5B). Cleavage reactions were then performed inReaction (R) buffer (10 mM Tris-HCl, pH 7.5 at 37° C., 100 mM NaCl and 1mM DTT, 10 mM MgCl2) under various conditions. For experiments assayingthe metal co-factor, the magnesium in buffer R was replaced with eithercobalt (Co²⁺), manganese (Mn²⁺), or nickel (Ni²⁺). Supercoileddouble-stranded plasmid DNA cleavage was assessed by examining the ratioof supercoiled (SC), nicked open circle (OC), and full length linearized(FLL) products. For linear double-stranded plasmid DNA substrates, thefraction of un-cleaved products relative to the smaller cleaved productswas used to calculate cleavage efficiency.

Example 7: Optimization of Cas-Alpha Components for Eukaryotic GenomeEditing and Manipulation

In this Example, methods to optimize Cas-alpha endonuclease and guideRNA expression cassettes or purified components for delivery intoeukaryotic cells are described.

In one method, to confer efficient expression in eukaryotic cells, thenovel Cas endonuclease gene, cas-alpha, was codon optimized per standardtechniques known in the art and optionally an intron was introduced inorder to eliminate its expression in E. coli or Agrobacterium (used forplant transformation). For use in Zea mays, the potato ST-LS1 intron 2(SEQ ID NO: 106) was used although other introns would work. Tofacilitate nuclear localization of the optimized Cas-alpha endonucleaseprotein in eukaryotic cells, a nucleotide sequence encoding the Simianvirus 40 (SV40) monopartite nuclear localization signal (NLS) (SEQ IDNO: 107) may be added to either the 5′, 3′, or both 5′ and 3′ ends.Other NLSs can also be used. For example, in human cell cultureexperiments, a sequence encoding the bi-partite NLS from nucleoplasmin(Nuc) (SEQ ID NO: 108) was optionally appended to the 3′ end of thehuman codon optimized gene. The nucleotide sequences of the differentmaize optimized Cas-alpha endonuclease gene and nuclear localizationsignal variants, were then operably linked to a promoter (ubiquitin(UBI) promoter (SEQ ID NO: 109) for maize expression constructs andchicken β-actin promoter (SEQ ID NO: 110) for human cell cultureexpression constructs) and optionally an enhancer (for example thecytomegalovirus (CMV) enhancer (SEQ ID NO: 111) for human cell genomeediting) and suitable terminator by standard molecular biologicaltechniques. To further enhance expression, a 5′ untranslated region(UTR) (for example but not limited to the maize UBI 5′ UTR (SEQ ID NO:112) for Zea mays genome editing) and additional introns (for examplethe UBI Zea mays intron 1 (SEQ ID NO: 113) for maize genome editing anda synthetic “hybrid” intron (SEQ ID NO: 114) for human cell genomeediting) can be included. Additionally, reduced (for example but notlimited to a ROX3 promoter (SEQ ID NO:136) for Saccharomyces cerevisiaegenome editing) or controlled (for example but not limited to a GALpromoter (SEQ ID NO:137) for Saccharomyces cerevisiae genome editing)expression might be desirable. Examples of the eukaryotic cell optimizedDNA expression constructs are illustrated in FIG. 10A-D.

The Cas-alpha endonuclease is directed by small RNAs (referred to hereinas guide RNAs) to cleave double-stranded DNA. These guide RNAs comprisea sequence that aids recognition by Cas-alpha (referred to as Cas-alpharecognition domain) and a sequence that serves to direct Cas-alphacleavage by base pairing with one strand of the DNA target site(Cas-alpha variable targeting domain). To transcribe small RNAsnecessary for directing Cas-alpha endonuclease cleavage activity inmaize cells, a U6 polymerase III promoter (SEQ ID NO: 115) andterminator (TTTTTTTT) are isolated from maize and operably fused to theends of DNA sequences that upon transcription would result in a suitableguide RNA for Cas-alpha. Alternatively, for HEK293 cells, a U6 promoterfrom the human genome (SEQ ID NO: 116) is isolated and used to driveguide RNA expression and a linear fragment containing without a U6terminator is utilized. To promote optimal transcription of the guideRNA from the U6 polymerase III promoters a G nucleotide is added to the5′ end of the sequence to be transcribed. Polymerase II promoters (forexample but not limited to those listed for Cas-alpha endonucleaseexpression) in combination with a ribozyme motif (Gao, Y. et al. (2014)J Integr Plant Biol. 56:343-349)), RNase P and Z cleavage sites (Xie, K.et al. (2015) Proc. Natl. Acad. Sci. USA. 112:3570-3575), and/or Csy4(Cas6 or CasE) ribonuclease recognition site (Tsai, S. Q. et al. (2014)Nat Biotechnol. 32:569-576.) can also be used to express the guide RNA.Moreover, the RNA processing provided by these strategies can also beharnessed to express multiple guide RNAs from either a single polymeraseII or III promoter (Gao, Y. et al. (2014), Xie, K. et al. (2015), andTsai, S. Q. et al. (2014)). Examples of the eukaryotic optimizedCas-alpha guide RNA expression constructs are illustrated in FIG. 11A-D.

In another method, Cas-alpha endonuclease and guide RNAribonucleoprotein (RNP) complexes were prepared and delivered directlyinto the eukaryotic cell. To accomplish this, Cas-alpha genes, eithernative or E. coli codon optimized, were appended with sequences encodinga 6× histidine (His) (SEQ ID NO: 82) or streptavidin (strep II) (SEQ IDNO: 117) tag, a maltose binding protein (MBP) tag (SEQ ID NO: 83), atobacco etch virus cleavage site (TEV) (SEQ ID NO:84), and a NLS (eitherSEQ ID NO: 107 and 108) included either at the N- or C-terminal or bothN- and C-terminal ends of the cas-alpha gene (FIG. 12). Next, theresulting sequences were synthesized (GenScript) into an arabinoseinducible E. coli expression cassette (pBAD24). Example of the resultingengineered genes are shown in FIG. 12. Then, Cas-alpha protein wasrecombinantly expressed in E. coli (for example but not limited toArcticExpress (DE3) (ThermoFisher Scientific) and purified bychromatography using methods known in the art. The tags (His, strep II,and MBP) were optionally removed using the TEV protease (ThermoFisherScientific).

Next, Cas-alpha guide RNAs were synthesized in vitro using T7polymerase. Linear DNA (synthesized as overlapping oligos (IDT) and thenconverted into double stranded DNA by PCR or synthesized (GenScript) andthen amplified by PCR) encoding the sgRNA was used as template.

Finally, RNP complexes were prepared by incubating purified Cas-alphaprotein with the guide RNA in complex assembly (CA) buffer (10 mMTris-HCl, pH 7.5 at 37° C., 100 mM NaCl and 1 mM DTT) and delivered intothe eukaryotic cell.

Example 8: Transformation of Optimized Cas-Alpha System Components forEukaryotic Genome Editing and Manipulation

In this example, methods for introducing a novel Class 2 endonuclease(Cas-alpha) and associated guide polynucleotide(s) into eukaryotic cellsfor genome editing and manipulation are described.

Zea mays Transformation

Particle-Mediated Delivery of DNA Expression Cassettes

Particle gun transformation of Hi-Type II 8 to 10-day-old immature maizeembryos (IMEs) in the presence of BBM and WUS2 genes was carried-out asdescribed in Svitashev et al. (2015) Plant Physiology. 169:931-945.Briefly, DNA expression cassettes were co-precipitated onto 0.6 μM(average size) gold particles utilizing TransIT-2020. Next, the DNAcoated gold particles were pelleted by centrifugation, washed withabsolute ethanol and re-dispersed by sonication. Following sonication,10 μl of the DNA coated gold particles were loaded onto a macrocarrierand air dried. Next, biolistic transformation was performed using aPDS-1000/He Gun (Bio-Rad) with a 425 pound per square inch rupture disc.Since particle gun transformation can be highly variable, a visualmarker DNA expression cassette encoding a yellow fluorescent protein(YFP) was also co-delivered to aid in the selection of evenlytransformed IMEs and each treatment was performed in triplicate. Todetermine the plant transformation culture conditions optimal forCas-alpha binding or mutational activity, transformed IMEs is incubatedat 28° C. for 48 hours, or at a range of temperatures lower or higherthan 28° C. to establish the temperature optimum for Cas-alpha genomeediting.

Particle-Mediated Ribonucleoprotein Delivery

Cas-alpha and associated guide polynucleotide(s) ribonucleoprotein (RNP)complex(es) can be delivered by particle gun transformation as describedin Svitashev, S. et al. (2016) Nat. Commun. 7:13274. Briefly, RNPs (andoptionally DNA expression) are precipitated onto 0.6 mm (averagediameter) gold particles (Bio-Rad) using a water soluble cationic lipidTransIT-2020 (Minis) as follows: 50 ml of gold particles (watersuspension of 10 mg/ml) and 2 ml of TransIT-2020 water solution areadded to the premixed RNPs (and optionally DNA expression vectors),mixed gently, and incubated on ice for 10 min. RNP/DNA-coated goldparticles are then pelleted in a microfuge at 8,000 g for 30 s andsupernatant is removed. The pellet is then resuspended in 50 ml ofsterile water by brief sonication. Immediately after sonication, coatedgold particles are loaded onto a microcarrier (10 ml each) and allowedto air dry. Immature maize embryos, 8-10 days after pollination, arethen bombarded using a PDS-1000/He Gun (Bio-Rad) with a rupture pressureof 425 pounds per inch square. Post-bombardment culture, selection, andplant regeneration are performed using methods known in the art.

Agrobacterium-Mediated Transformation

Agrobacterium-mediated transformation is performed essentially asdescribed in Djukanovic et al. (2006) Plant Biotech J 4:345-57. Briefly,10-12 day old immature embryos (0.8-2.5 mm in size) are dissected fromsterilized kernels and placed into liquid medium (4.0 g/L N6 Basal Salts(Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/Lthiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/L L-proline, 68.5 g/L sucrose,36.0 g/L glucose, pH 5.2). After embryo collection, the medium isreplaced with 1 ml Agrobacterium at a concentration of 0.35-0.45 OD550.Maize embryos are incubated with Agrobacterium for 5 min at roomtemperature, then the mixture is poured onto a media plate containing4.0 g/L N6 Basal Salts (Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix(Sigma E-1511), 1.0 mg/L thiamine HCl, 1.5 mg/L 2, 4-D, 0.690 g/LL-proline, 30.0 g/L sucrose, 0.85 mg/L silver nitrate, 0.1 nMacetosyringone, and 3.0 g/L Gelrite, pH 5.8. Embryos are incubated axisdown, in the dark for 3 days at 20° C., then incubated 4 days in thedark at 28° C. at which time they may be harvested for DNA extraction.

In another variation for stable transformation, the embryos are thentransferred onto new media plates containing 4.0 g/L N6 Basal Salts(Sigma C-1416), 1.0 ml/L Eriksson's Vitamin Mix (Sigma E-1511), 1.0 mg/Lthiamine HCl, 1.5 mg/L 2, 4-D, 0.69 g/L L-proline, 30.0 g/L sucrose, 0.5g/L MES buffer, 0.85 mg/L silver nitrate, 3.0 mg/L Bialaphos, 100 mg/Lcarbenicillin, and 6.0 g/L agar, pH 5.8. Embryos are subcultured everythree weeks until transgenic events are identified. Somaticembryogenesis is induced by transferring a small amount of tissue ontoregeneration medium (4.3 g/L MS salts (Gibco 11117), 5.0 ml/L MSVitamins Stock Solution, 100 mg/L myo-inositol, 0.1 uM ABA, 1 mg/L IAA,0.5 mg/L zeatin, 60.0 g/L sucrose, 1.5 mg/L Bialaphos, 100 mg/Lcarbenicillin, 3.0 g/L Gelrite, pH 5.6) and incubation in the dark fortwo weeks at 28° C. All material with visible shoots and roots aretransferred onto media containing 4.3 g/L MS salts (Gibco 11117), 5.0ml/L MS Vitamins Stock Solution, 100 mg/L myo-inositol, 40.0 g/Lsucrose, 1.5 g/L Gelrite, pH 5.6, and incubated under artificial lightat 28° C. One week later, plantlets are moved into glass tubescontaining the same medium and grown until they were sampled and/ortransplanted into soil.

HEK293 Transformation Cell Culture Lipofection

HEK293 (ATCC) cells were cultured in DMEM (Gibco) with 10% FBS (Gibco)and penicillin/streptomycin (Gibco) at 37° C. in 5% CO₂. A day prior totransfection cells were seeded in 96-well plates at 3.6×10⁴ density.NLS-tagged Cas-alpha RNP complex was assembled by mixing 20 pmol ofpurified protein with 20 pmol of sgRNA in 25 μl Opti-MEM (Gibco) andincubated at room temperature for 30 min. After complex assembly 25 μlof Opti-MEM, containing 1.2 μl Lipofectamine 3000 (Thermo FisherScientific) was added and the mixture was incubated for additional 15min at room temperature before transfection of the cells. Genomic DNAwas extracted 72 h after transfection using QuickExtract DNA ExtractionSolution (Lucigen) and regions surrounding target sites evaluate for thepresence of mutations indicative of DNA double strand break and repair.

Cell Culture Electroporation

Cas9 RNPs were electroporated into HEK293 (ATCC Cat # CRL-1573) cellsusing the Lonza 4D-Nucleofector System and the SF Cell Line4D-Nucleofector® X Kit (Lonza). For each electroporation, RNPs wereformed by incubating 100 pmoles of sgRNA with 50 pmoles of Cas9 proteinin nucleofector solution in a volume of 17 μL at room temperature for 20minutes. HEK293 cells were released from culture vessels using TrypLE™Express Enzyme 1× (ThermoFisher) washed with 1×PBS without Ca++ or Mg++(ThermoFisher) and counted using a LUNA™ Automated Cell Counter (LogosBiosystems). For each electroporation, 1×10{circumflex over ( )}5 livecells were resuspended in 9 μL electroporation solution. Cells and RNPwere mixed and transferred to one well of a 16-well strip andelectroporated using the CM-130 program. 754, of pre-warmed culture wasadded to each well and 10 μL of the resultant resuspended cells weredispensed into a well of a 96-well culture vessel containing 125 μL ofpre-warmed culture medium. Electroporated cells were incubated at 37°C., 5% CO2 in a humidified incubator for 48-96 hours before analysis ofgenome editing.

Saccharomyces cerevisiae Transformation

Several methods (lithium acetate, polyethylene glycol (PEG), heat shock,electroporation, biolistic, and others) can be used to transform S.cerevisiae (Kawai, S. et al. (2010) Bioengineered Bugs. 1:395-403). Herewe used an approach similar to a lithium cation-based method using theFrozen-EZ yeast Transformation II kit (Zymo Research, T2001). Per themanufacture's instruction, S. cerevisiae competent cells were produced.This was accomplished by growing S. cerevisiae (BY4742 (Baker, C. et al.(1998) Yeast. 14:115-132) (ATCC)) in yeast extract-peptone-dextrose(YPD) (Gibco) to mid-log phase corresponding to an OD 600 nm of 0.8-1.0.Next, the cells were pelleted by centrifugation (500×g for 4 minutes),media decanted, and the pellet gently washed with 10 ml of EZ 1 solutionspinning down the cells again prior to removing the wash solution. Next,the cells were resuspended in 1 ml of EZ 2 solution. The resultingcompetent cells were then aliquoted and either stored at −70° C. or usedin the next step. Transformation was performed next by adding 0.5-1 μg(in less than 5 μl) of Cas-alpha and guide RNA DNA expression cassettesto 50 ul of competent cells. Optionally, double stranded DNA repairtemplate with homology flanking the expected Cas-alpha double strandbreak site was also included (0.5 ul at 50 μM). After gently mixing inthe DNA, 500 μl of EZ 3 solution was added. Next, cells were incubatedat 30° C. for 60-90 min. flicking or vortexing the cells 3-4 times overthe duration of the incubation. After transformation, cells weregrown-out in YPD for ˜3 hrs, pelleted, washed once with 1 ml of sterilewater, resuspended in 1 ml of sterile water, and then ˜200 μl platedonto selective media (for example but not limited to Synthetic mediumminus histidine (SC-HIS)).

Example 9: Functional Formation of an Optimized Cas-Alpha/GuidePolynucleotide Complex in Eukaryotic Cells

In this example, a method for examining the functional formation of anovel Class 2 endonuclease (Cas-alpha) and associated guided RNA(s)(polynucleotide(s)) complex in eukaryotic cells are described.

The functional formation of a novel Class 2 endonuclease (Cas-alpha) andguide RNA(s) complex in eukaryotic cells was monitored by examining oneor more different chromosomal DNA target sequences for the presence ofinsertion and deletion (indel) mutations indicative of DNA target sitedouble stranded cleavage and cellular repair. This was carried-out bytargeted deep sequencing as described in described in Karvelis, T. etal. (2015) Genome Biology. 16:253 (Methods Section: in planta mutationdetection) or other equivalent methods devised to detect alterations inDNA. Briefly, for Zea mays, the 20-30 most evenly transformed immatureembryos (IEs), based on their fluorescence, were harvested two daysafter transformation for each experiment. Next, total genomic DNA wasextracted and the region surrounding the intended target site was PCRamplified with Phusion® HighFidelity PCR Master Mix (New EnglandBiolabs, M0531L) adding on the sequences necessary for amplicon-specificbarcodes and Illumina sequencing using “tailed” primers through tworounds of PCR and deep sequenced. The resulting reads were then examinedfor the presence of mutations at the expected site of cleavage bycomparison to control experiments where the small RNA transcriptionalcassette was omitted from the transformation. Sequence reads containingputative indels where further validated as true mutations by confirmingtheir absence in the control datasets.

For Saccharomyces cerevisiae, a similar approach was applied exceptcolonies exhibiting a red cellular phenotype resulting from thedisruption of the ade2 gene (Ugolini et al. (1996) Curr. Genet.30:485-492) were selected prior to DNA extraction, PCR amplification,and Illumina deep sequencing.

For HEK293, a similar process was performed except the cell culture washarvested 72 hours after transformation.

As illustrated in FIG. 13 and shown in Table 16, particle gunexperiments delivering Cas-alpha DNA expression constructs into Zea maysIEs yielded predominantly deletion mutations at and encompassing thechromosomal DNA target sites for the Cas-alpha4 and guide RNA complex.In these experiments, a maize codon optimized cas-alpha4 gene (SEQ IDNO: 235) configured for expression as shown in FIG. 10B (except tosequence encoding the SV40 NLS was added in-frame at the 3′ end of thegene) was used. sgRNAs (Table 19) with a 20 nt region capable of basepairing with a chromosomal DNA target sequence immediately adjacent to asuitable PAM for Cas-alpha4 (5′-TTTR-3′, where R represents either A orG residues; see Table 7) were expressed from a Zea mays U6 promoter asillustrated in FIG. 11B. In this instance, two guide RNAs were used todirect Cas-alpha4 cleavage at two target sites in the Zea maysLiguleless locus.

TABLE 16 Cas-alpha endonucleases generate targeted double strand breaksin plant cell genomic DNA Mutations were found at plant cell genomictarget sites as a result of Cas-alpha4 endonuclease target DNA cleavageand double strand break repair. All mutations displayed positive readcounts, and none were found in the negative control samples. Found inSequence reads SEQ ID Negative Read recovered NO: Control? Count WTReference 120 Yes 583996 Negative Control — — 0 Mutation 1 121 No 4Mutation 2 122 No 2 Mutation 3 123 No 2 Mutation 4 124 No 2

Furthermore, as illustrated in FIGS. 18A and B and shown in Table 17,particle gun experiments delivering Cas-alpha10 DNA expressionconstructs into Zea mays IEs resulted in the recovery of targeteddeletions. In these experiments, a maize codon optimized cas-alpha10gene (SEQ ID NO: 236) configured for expression as shown in FIG. 10B wasused (except a sequence encoding the SV40 NLS was added in-frame at the3′ end of the gene). sgRNAs (Table 19) with a 20 nt region capable ofbase pairing with a chromosomal DNA target sequence immediately adjacentto a suitable PAM for Cas-alpha10 were expressed from a Zea mays U6promoter as illustrated in FIG. 11B. A transgenic construct drivingexpression of a plant selectable marker, neomycin phosphotransferase(nptII), stably inserted into the maize genome was targeted for cleavagewith Cas-alpha10 (5′-TTC-3′; Table 16). As shown in FIG. 18A and Table17, deletions not found in the controls (experiments setup omitting thesgRNA expression cassette) were recovered that originated within orspanned the expected site of cleavage. To confirm our findings, a singlenon-transgenic chromosomal DNA target within the fifth exon of the ms26gene (Chr1:14,702,638-14,702,654 (Maize B73 RefGen_4 (Jiao, Y. et al.(2017) Nature. 546:524-527)) was also targeted for cleavage (using sgRNA10.25.ms26 in Table 19). Like the nptII target, this site also producedtargeted deletions at or near the nuclease cut site (FIG. 18A, 18B andTable 17).

TABLE 17 Cas-alpha10 generates targeted double strand breaks in plantcell genomic DNA Mutations were found at plant cell genomic target sitesas a result of Cas-alpha10 endonuclease target DNA cleavage and doublestrand break repair. All mutations displayed positive read counts, andnone were found in the negative control samples. Found in Zea maysSequence reads SEQ ID Negative Read target site recovered NO: Controls?Count nptII WT Reference 144 Yes 155252 Negative Control — — 0 Mutation1 145 No 15 Mutation 2 146 No 13 Mutation 3 147 No 11 Mutation 4 148 No10 Mutation 5 149 No 10 Mutation 6 150 No 8 Mutation 7 151 No 8 Mutation8 152 No 8 Mutation 9 153 No 8 Mutation 10 154 No 8 Mutation 11 155 No 6Mutation 12 156 No 5 Mutation 13 157 No 5 Mutation 14 158 No 5 Mutation15 159 No 5 Mutation 16 160 No 4 Mutation 17 161 No 2 Mutation 18 162 No2 Mutation 19 163 No 2 ms26 WT Reference 164 Yes 581646 Negative Control— — 0 Mutation 1 165 No 57 Mutation 2 166 No 26 Mutation 3 167 No 24Mutation 4 168 No 13 Mutation 5 169 No 5

Target DNA cleavage and repair was also observed in Saccharomycescerevisiae (FIGS. 19A-C). Here, an exogenously supplied DNA repairtemplate (double stranded) with homology flanking a Cas-alpha10 targetsite was used to introduce one or two premature stop codons (dependingon the DNA repair outcome) in the ade2 gene following a Cas-alpha10induced double strand break (DSB) (FIG. 19A). Additionally, to avoidtargeting of the repair template, it also contained a T to A change inthe PAM region for Cas-alpha10. As shown, in FIG. 19B, a red cellularphenotype indicative of ade2 gene disruption was recovered when both therepair template and Cas-alpha10 and sgRNA expression constructs weretransformed. The Cas-alpha10 expression construct was configured asshown in FIG. 10C using a yeast codon optimized gene (SEQ ID NO: 137).The ade2 targeting sgRNA (Table 19) was expressed from a SNR52 promoterusing flanking HH and HDV ribozymes (FIG. 11C). Sequencing of theCas-alpha10 ade2 gene target site confirmed the introduction of at leastone stop codon in 3 independent red colonies (FIG. 19C). Additionally,only the changes in the repair template closest to the Cas-alpha10 siteof cleavage were incorporated providing further evidence for the repairof a Cas-alpha10 induced DSB (FIG. 19C). Moreover, this repair outcomesuggests that only one or two mismatches towards the distal end of theguide RNA target was enough to abolish cleavage activity (since no othermutations were recovered), altogether, indicating that Cas-alphanucleases provide excellent guide RNA-DNA target recognitionspecificity. To confirm that Cas-alpha10 was absolutely required for theoutcome, control experiments delivering the DNA repair template alonewere assembled. They produced only white colonies, further validatingthe ability of Cas-alpha10 (and guide RNA) to recognize and cleave achromosomal DNA target site as measured here by homology-directedrepair.

DNA cleavage and repair of HEK293 chromosomal targets also resulted indeletion mutations (FIGS. 14A and B and Table 18). Transformationexperiments performed both with DNA expression cassettes (see FIGS. 10Aand 11A) and directly with eukaryotic engineered Cas-alpha4 sgRNAribonucleoprotein (RNP) complexes yielded mutations. In all, mutationswere recovered from two HEK293 genomic targets, VEGFA2 and 3 (FIGS. 14Aand B).

TABLE 18 Cas-alpha endonucleases generates targeted double strand breaksin animal cell genomic DNA Mutations were found at animal cell genomictarget sites as a result of Cas-alpha endonuclease target DNA cleavageand double strand break repair. All mutations displayed positive readcounts, and none were found in the negative control samples. Found inRead Delivery Controls? Count Ribonucleo- VEGFA 2 protein Wt Reference385293 (RNP) Negative Control 0 Mutation 1 No 442 VEGFA 2 Wt Reference260251 Negative Control 0 Mutation 1 No 54 VEGFA 2 Wt Reference 265110Negative Control 0 Mutation 1 No 186 Mutation 2 No 95 Mutation 3 No 84VEGFA 3 Wt Reference 942942 Negative Control 0 Mutation 1 No 164 DNAVEGFA 3 Expression Wt Reference 160628

Negative Control 0 Mutation 1 No 58 VEGFA 3 Wt Reference 212590 NegativeControl 0 Mutation 1 No 45

indicates data missing or illegible when filed

TABLE 19 Cas-alpha single guide RNAs producing targeted mutagenesis inZea mays, Saccharomyces cerevisiae, and Homo sapiens (HEK293) cellssgRNA sgRNA Target Complete sgRNA “Backbone” Sequence (“backbone” + Cas-(SEQ ID (SEQ ID Target Sequence) alpha Name NO.) NO.) (SEQ ID NO.) 4Liguleless 2 238 240 247 4 Liguleless 3 238 241 248 10 nptII 239 242 24910 ms26 239 243 250 10 ade2 239 244 251 4 VEGFA2 238 245 252 4 VEGFA3238 246 253

Mutations due to Cas-alpha double strand break cleavage and repair at agenomic DNA target site were recovered in plant, yeast, and animalcells, with examples of using recombinant DNA constructs as well asribonucleoprotein delivery. These data present the first evidence ofCas-alpha guide polynucleotide complex formation and cleavage activityin eukaryotic cells, plant (Zea mays), yeast (Saccharomyces cerevisiae),and animal (Homo sapiens) cells.

Example 10: Double Strand DNA Cleavage in Prokaryotic Cell Assays

In this example, a method for examining the functional formation of anovel Class 2 endonuclease (Cas-alpha) and associated guided RNA(s)(polynucleotide(s)) complex in heterologous prokaryotic cells aredescribed.

As shown in FIG. 17A, one method to assess Cas-alpha double stranded DNAtarget cleavage is to examine its ability to interfere with plasmid DNAtransformation in E. coli cells (Burstein, D. et al. (2017) Nature.542:237-241). Here, a double stranded plasmid DNA comprising aselectable marker (for example but not limited to Ampicillin) and aCas-alpha target site (a region capable of base pairing with the CRISPRRNA that is in the vicinity of a protospacer adjacent motif (PAM)) istransformed into E. coli (ArcticExpress DE3 or equivalent) that containsa Cas-alpha endonuclease and guide RNA expression cassette, by methodsknown in the art (for example but not limited to electroporation). Inthe absence of double stranded DNA target cleavage, many cellscontaining the plasmid and antibiotic resistance marker are recovered bygrowth on selective media. In contrast, double stranded DNA targetcleavage of the in-coming plasmid DNA results in a reduction orinterference in the recovery of resistant cells.

To assess the dsDNA cleavage activity of Cas-alpha2, 3, 6, 7, 8, 9, 10,and 11, plasmid DNA interference experiments were assembled in E. colicells. Experiments setup with plasmids that didn't contain a Cas-alphatarget site, “no target”, provided a baseline for transformationefficiency. Also, interference experiments were performed with andwithout IPTG (0.5 mM) to examine target cleavage under differentCas-alpha endonuclease and guide RNA expression conditions. 100 ng ofeither “target” or “no target” plasmid DNA was transformed into ArcticExpress (DE3) cell lines containing IPTG inducible Cas-alphaendonuclease and guide RNA expression cassettes (e.g. R-657).Transformations were diluted in 10-fold increments, spotted on selectivemedia, grown overnight at 37° C., and inspected for bacterial colonygrowth.

FIGS. 17B-17E show the results for Cas-alphas 2, 3, 6, 7, 8, 9, 10, and11. Cas-alpha3 and 11 (FIGS. 17B and E) were cytotoxic upon induction ofexpression as evident by a reduced recovery of transformants in both “notarget” and “target” experiments and Cas-alpha2 and 6 failed to exhibitany impact on plasmid transformation (FIGS. 17B and C). This can becontrasted with Cas-alpha7 and 9 that provided weak interferenceactivity (FIGS. 17C and D) and Cas-alpha8 and 10 that robustly decreasedthe number of “target” transformed colonies FIGS. 17D and E.

Taken together, this illustrates that some but not all Cas-alphaendonucleases and guide RNAs function to recognize and cleave dsDNAtargets in a heterologous prokaryotic cellular environment.

Example 11: Cas-Alpha Phylogenetic Analysis

In this Example, methods for evaluating the phylogenetic relationship ofa novel group of class 2 CRISPR (Clustered Regularly Interspaced ShortPalindromic Repeat)-Cas (CRISPR associated) endonucleases, Cas-alpha,are described.

To identify distant relatives, two iterations of PSI-BLAST wereperformed with Cas-alpha 1-11 selecting only those alignments containingat least 70% full-length coverage for the construction ofposition-specific scoring matrices (PSSMs) between rounds of PSI-BLAST.Next, only those proteins that were encoded adjacent to a CRISPR array(as detected by MinCED) were selected resulting in the identification of118 additional Cas-alpha endonucleases (SEQ ID NO: 254-371) ranging insize from 327-777 amino acids. Phylogenetic analysis (Maximum Likelihoodmethod and JTT matrix-based model (Jones, D. T. et al., (1992) ComputerApplications in the Biosciences 8: 275-282) using MEGA software (version10.0.5) (Kumar, S. et al. (2018) Molecular Biology and Evolution.35:1547-1549)) was then performed. It showed the formation of threedistinct groups (I, II, and III.) of Cas-alpha nucleases with themajority coming from three lineages of microorganisms, CandidatusArchaea, Clostridia, and Bacilli (FIG. 20). Those loci that also encodedCRISPR-Cas adaption genes (Cas1, Cas2, and optionally Cas4) were onlyassociated with Cas-alpha proteins from Archaea. Other bacteria whereCas-alpha nucleases were identified included organisms belonging toAquificae, Deltaproteobacteria, Bacteroidetes, Candidate Levybacterium,Negativicutes, and Flavobacteriia (FIG. 20). Additionally, the topologyof the cladogram only partially matched the microorganism from which theCas-alpha endonucleases were identified. Most of the discrepancy camefrom group III that is present in both Bacilli and Clostrida, suggestinghorizontal transfer between these two classes of microorganisms (FIG.20).

Example 12: Cas-Alpha RNA-Guided DNA Integrase

In this Example, a Cas-alpha endonuclease and guide polynucleotide incomplex with a transposase (for example but not limited to TnpA) can beutilized to site-specifically insert a DNA payload.

Tn7-like genetic mobile elements have captured CRISPR-associated (Cas)proteins (Peters, J. et al. (2017) Proc. Natl. Acad. Sci. USA.114:E7358-E7366) and evolved RNA-guided based mechanisms to copythemselves into new locations and offer to advance genome editingapproaches that rely on the insertion of DNA (for examples but notlimited to cis- or trans-genes) at specific sites (Strecker, J. et al.(2019) Science. 365:48-53 and Klompe, S. et al. (2019) Nature.571:219-225). Here, we find that transposase (Tnp) proteins belonging toIS200/IS605 and IS4 mobile elements are encoded adjacent to someCas-alpha endonucleases (FIG. 21A). Taken together, this suggests thatCas-alpha endonucleases can be harnessed to function as part of atransposase complex capable of programmable DNA integration (FIG. 21B).

1. A synthetic composition comprising: (a) a Cas endonuclease at least90% identical to SEQ ID NO: 37, or a functional fragment or variantthereof, wherein the Cas endonuclease comprises the following amino acidmotifs: GxxxG, ExL, Cx_(n)C, and Cx_(n)(C or H); wherein G=Glycine,E=Glutamate, C=Cysteine, H=Histidine, x=any amino acid and wherein n=aninteger between 0 and 11; (b) a target double-stranded DNApolynucleotide, wherein the target double-stranded DNA polynucleotide isheterologous to the source of the Cas endonuclease, and (c) a guidepolynucleotide comprising a variable targeting domain that comprises aregion of complementarity to the target double-stranded DNApolynucleotide; wherein the Cas endonuclease recognizes a PAM sequenceon the target double-stranded DNA polynucleotide, wherein the guidepolynucleotide and the Cas endonuclease form a complex that binds to thetarget double-stranded DNA polynucleotide.
 2. The synthetic compositionof claim 1, wherein the Cas endonuclease is provided as a polynucleotideencoding the Cas endonuclease.
 3. The synthetic composition of claim 1,wherein the Cas endonuclease cleaves the double-stranded DNApolynucleotide.
 4. The synthetic composition of claim 1, furthercomprising a heterologous polynucleotide.
 5. The synthetic compositionof claim 4, wherein the heterologous polynucleotide is an expressionelement.
 6. The synthetic composition of claim 4, wherein theheterologous polynucleotide is a transgene.
 7. The synthetic compositionof claim 4, wherein the heterologous polynucleotide is a donor DNAmolecule.
 8. The synthetic composition of claim 4, wherein theheterologous polynucleotide is a polynucleotide modification template.9. The synthetic composition of claim 1, wherein the CRISPR-Casendonuclease is catalytically inactive.
 10. The synthetic composition ofclaim 1, further comprising a deaminase.
 11. The synthetic compositionof claim 1, wherein the Cas endonuclease is part of a fusion protein.12. The synthetic composition of claim 11, wherein the fusion proteinfurther comprises a heterologous nuclease domain.
 13. The syntheticcomposition of claim 1, further comprising a eukaryotic cell.
 14. Thesynthetic composition of claim 13, wherein the eukaryotic cell is aplant cell, an animal cell, or a fungal cell.
 15. The syntheticcomposition of claim 14, wherein the plant cell is a monocot cell or adicot cell.
 16. The synthetic composition of claim 14, wherein the plantcell is from an organism selected from the group consisting of: maize,soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye,barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa,sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.17. A polynucleotide encoding the Cas endonuclease of the syntheticcomposition of claim 1, further comprising at least one heterologouspolynucleotide.
 18. (canceled)
 19. The polynucleotide of claim 17,wherein the heterologous polynucleotide is an expression element. 20.The polynucleotide of claim 17, wherein the heterologous polynucleotideis a gene.
 21. The synthetic composition of claim 1, wherein at leastone component is attached to a solid matrix.
 22. A method of introducinga targeted edit in a target polynucleotide, comprising providing aheterologous composition comprising: (a) a Cas endonuclease comprising aCas endonuclease at least 90% identical to SEQ ID NO:37, or a functionalfragment or variant thereof, wherein the Cas endonuclease comprises thefollowing amino acid motifs: GxxxG, ExL, Cx_(n)C, and Cx_(n)(C or H);wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, x=any aminoacid and wherein n=an integer between 0 and 11, wherein the Casendonuclease recognizes a PAM sequence on the target polynucleotide; and(b) a guide polynucleotide comprising a variable targeting domain thatis substantially complementary to a portion of the targetpolynucleotide, wherein the guide polynucleotide and Cas-alphaendonuclease form a complex that can recognize and bind to the targetpolynucleotide.
 23. The method of claim 22, further comprising a cell,the method further comprising introducing at least one nucleotidemodification in the genome of at least one cell of the organism ascompared to the target sequence of the genome of the cell prior to theintroduction of the heterologous composition, further comprisingincubating the cell and generating a whole organism from the cell, andascertaining the presence of the at least one nucleotide modification inthe genome of at least one cell of the organism as compared to thetarget sequence of the genome of the cell prior to the introduction ofthe heterologous composition.
 24. A progeny of the organism obtained bythe method of claim 23, wherein the progeny retains the at least onenucleotide modification in at least one cell.
 25. The method of claim22, wherein the cell is a eukaryotic cell.
 26. The method of claim 25,wherein the eukaryotic cell is derived or obtained from an animal, afungus, or a plant.
 27. The method of claim 26, wherein the plant is amonocot or a dicot.
 28. The method of claim 26, wherein the plant isselected from the group consisting of: maize, soybean, cotton, wheat,canola, oilseed rape, sorghum, rice, rye, barley, millet, oats,sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut,potato, Arabidopsis, safflower, and tomato.
 29. The method of claim 22,further comprising introducing a heterologous polynucleotide
 30. Themethod of claim 29, wherein the heterologous polynucleotide is either adonor DNA molecule, or a polynucleotide modification template thatcomprises a sequence at least 50% identical to a sequence in the cell.